Mycobacterium avium subsp. paratuberculosis (MAP) has a high degree of resistance to chemical and physical procedures frequently used for the elimination of other bacteria. Recently, a method for the determination of viability by exposure of MAP to propidium monoazide (PMA) and subsequent real time quantitative PCR (qPCR) was established and found to be comparable with culture. The aim of this study was to apply the PMA qPCR method to determine the impact of increasing concentration or time and repeated cycles of the application of selected disinfectants on MAP viability. Different MAP isolates responded to the same type of stress in different ways. The laboratory strain CAPM 6381 had the highest tolerance, while the 8819 low-passage field isolate was the most sensitive. Ultraviolet exposure caused only a partial reduction in MAP viability; all MAP isolates were relatively resistant to chlorine. Only the application of peracetic acid led to the total elimination of MAP. Repeated application of the treatments resulted in more significant decreases in MAP viability compared to single increases in the concentration or time of exposure to the disinfectant.

• Klíčová slova
• Disinfection, Mycobacterium avium subsp. paratuberculosis, Propidium monoazide quantitative PCR, Viability,
• MeSH
• azidy * MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chlor farmakologie   MeSH
• dezinficiencia farmakologie   MeSH
• kvantitativní polymerázová řetězová reakce veterinární   MeSH
• kyselina peroctová farmakologie   MeSH
• mikrobiální viabilita účinky léků   MeSH
• Mycobacterium avium subsp. paratuberculosis účinky léků   MeSH
• propidium analogy a deriváty   MeSH
• ultrafialové záření MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy * MeSH
• bakteriální proteiny MeSH
• chlor MeSH
• dezinficiencia MeSH
• kyselina peroctová MeSH
• propidium monoazide MeSH Prohlížeč
• propidium MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) is known to be a very slow-growing organism. The fact that cells typically need several weeks to form visible colonies severely compromises the suitability of plate counting for assessment of viable cell counts. This problem might be overcome by the application of fast molecular methods containing a viability component. We have evaluated a promising technology combining sample treatment with propidium monoazide (PMA) prior to DNA extraction for selective detection of cells with intact cell membranes with detection of sequence element F57 by quantitative PCR (F57 qPCR). Element F57 is unique for MAP and is not known to exist in any other bacterial species. Conditions of PMA treatment were optimised for MAP isolate 7082 using live and heat-killed cells and comparing different DNA extraction procedures. The subsequent successful application of the optimised protocol to four other MAP isolates of different origins suggested that the optimised protocol might be broadly applicable to different MAP strains. Furthermore, different equations were compared to use the data resulting from this technology to optimally predict the percentage of live MAP cells in mixtures containing both live and dead cells. The presented protocol holds promise to be used routinely for detecting MAP with intact cell membranes in research applications.

• MeSH
• azidy * MeSH
• bakteriální nálož MeSH
• bezpečnost potravin metody   MeSH
• buněčná membrána MeSH
• DNA bakterií izolace a purifikace   MeSH
• mikrobiální viabilita * MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   izolace a purifikace   MeSH
• polymerázová řetězová reakce metody   MeSH
• potravinářská mikrobiologie * MeSH
• propidium analogy a deriváty   MeSH
• vaječný žloutek mikrobiologie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy * MeSH
• DNA bakterií MeSH
• propidium monoazide MeSH Prohlížeč
• propidium MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) is a pathogenic bacterium causing the paratuberculosis, chronic and infectious disease common particularly in wild and domestic ruminants. Currently, culture techniques to detect viable MAP are still used most commonly, although these require a long incubation period. Consequently, a faster molecular method for assessing MAP cell viability based on cell membrane integrity was introduced consisting of sample treatment with the intercalation dye propidium monoazide (PMA) followed by quantitative PCR (qPCR). However, the PMA-qPCR assay is complicated by demanding procedures involving work in a darkroom and on ice. In this study, we therefore optimized a viability assay combining sample treatment with palladium (Pd) compounds as an alternative viability marker to PMA, which does not require such laborious procedures, with subsequent qPCR. The optimized Pd-qPCR conditions consisting of 90 min exposure to 30 µM bis(benzonitrile)dichloropalladium(II) or 30 µM palladium(II)acetate at 5 °C and using ultrapure water as a resuspension medium resulted in differences in quantification cycle (Cq) values between treated live and dead MAP cells of 8.5 and 7.9, respectively, corresponding to approximately 2.5 log units. In addition, Pd-qPCR proved to be superior to PMA-qPCR in distinguishing between live and dead MAP cells. The Pd-qPCR viability assay thus has the potential to replace time-consuming culture methods and demanding PMA-qPCR in the detection and quantification of viable MAP cells with possible application in food, feed, clinical and environmental samples.

• MeSH
• azidy farmakologie   MeSH
• biotest MeSH
• kvantitativní polymerázová řetězová reakce metody   MeSH
• mikrobiální viabilita MeSH
• Mycobacterium avium subsp. paratuberculosis * genetika   MeSH
• palladium farmakologie   MeSH
• paratuberkulóza * mikrobiologie   MeSH
• propidium farmakologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azidy MeSH
• palladium MeSH
• propidium MeSH

Antigen testing for SARS-CoV-2 (AGT) is generally considered inferior to RT-PCR testing in terms of sensitivity. However, little is known about the infectiousness of RT-PCR positive patients who pass undetected by AGT. In a screening setting for mildly symptomatic or asymptomatic patients with high COVID-19 prevalence (30-40%), 1141 patients were tested using one of five AGTs and RT-PCR. Where the results differed, virus viability in the samples was tested on cell culture (CV-1 cells). The test battery included AGTs by JOYSBIO, Assure Tech, SD Biosensor, VivaChek Biotech and NDFOS. Sensitivities of the ATGs compared to RT-PCR ranged from 42% to 76%. The best test yielded a 76% sensitivity, 97% specificity, 92% positive, and 89% negative predictive values, respectively. However, in the best performing ATG tests, almost 90% of samples with "false negative" AGT results contained no viable virus. Corrected on the virus viability, sensitivities grew to 81-97% and, with one exception, the tests yielded high specificities >96%. Performance characteristics of the best test after adjustment were 96% sensitivity, 97% specificity, 92% positive, and 99% negative predictive values (high prevalence population). We, therefore, believe that virus viability should be considered when assessing the AGT performance. Also, our results indicate that a well-performing antigen test could in a high-prevalence setting serve as an excellent tool for identifying patients shedding viable virus. We also propose that the high proportion of RT-PCR-positive samples containing no viable virus in the group of "false negatives" of the antigen test should be further investigated with the aim of possibly preventing needless isolation of such patients.

• Klíčová slova
• COVID-19, RT-PCR, SARS-CoV-2, antigen testing, infectiousness, viability,
• MeSH
• antigeny virové analýza   MeSH
• COVID-19 diagnóza   imunologie   MeSH
• dospělí MeSH
• falešně negativní reakce MeSH
• lidé středního věku MeSH
• lidé MeSH
• mikrobiální viabilita * MeSH
• plošný screening MeSH
• SARS-CoV-2 imunologie   MeSH
• senzitivita a specificita MeSH
• sérologické testy metody   MeSH
• testování na COVID-19 průkazem nukleové kyseliny MeSH
• testování na COVID-19 metody   MeSH
• Check Tag
• dospělí MeSH
• lidé středního věku MeSH
• lidé MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antigeny virové MeSH

Mycobacterium avium subsp. paratuberculosis (MAP) represents a slow-growing bacterium causing paratuberculosis, especially in domestic and wild ruminants. Until recently, the assessment of MAP viability relied mainly on cultivation, which is very time consuming and is unable to detect viable but non-culturable cells. Subsequently, viability PCR, a method combining sample treatment with the DNA-modifying agent ethidium monoazide (EMA) or propidium monoazide (PMA) and quantitative PCR (qPCR), was developed, enabling the selective detection of MAP cells with an intact cell membrane. However, this technology requires a laborious procedure involving the need to work in the dark and on ice. In our study, a method based on a combination of platinum compound treatment and qPCR, which does not require such a demanding procedure, was investigated to determine mycobacterial cell viability. The conditions of platinum compound treatment were optimized for the fast-growing mycobacterium M. smegmatis using live and heat-killed cells. The optimal conditions consisting of a single treatment with 100 μM cis-dichlorodiammine platinum(II) for 60 min at 5°C resulted in a difference in quantification cycle (Cq) values between live and dead membrane-compromised mycobacterial cells of about 6 Cq corresponding to about 2 log10 units. This optimized viability assay was eventually applied to MAP cells and demonstrated a better ability to distinguish between live and heat-killed mycobacteria as compared to PMA. The viability assay combining the Pt treatment with qPCR thereby proved to be a promising method for the enumeration of viable MAP cells in foodstuffs, environmental, and clinical samples which could replace the time-consuming cultivation or laborious procedures required when using PMA.

• Klíčová slova
• Mycobacterium avium subsp. paratuberculosis, live-dead discrimination, mycobacteria, platinum, propidium monoazide, qPCR, viability,
• Publikační typ
• časopisecké články 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

Cultivation-based assays represent the gold standard for the assessment of virus infectivity; however, they are time-consuming and not suitable for every virus type. Pre-treatment with platinum (Pt) compounds followed by real-time PCR has been shown to discriminate between infectious and non-infectious RNA viruses. This study examined the effect of Pt and palladium (Pd) compounds on enveloped DNA viruses, paying attention to two significant pathogens of livestock - bovine herpesvirus-1 (BoHV-1) and African swine fever virus (ASFV). Native or heat-treated BoHV-1 suspension was incubated with the spectrum of Pt/Pd compounds. Bis(benzonitrile)palladium(II) dichloride (BB-PdCl 2) and dichloro(1,5-cyclooctadiene) palladium(II) (PdCl 2-COD) produced the highest differences found between native and heat- -treated viruses. Optimized pre-treatment conditions (1 mM of Pd compound, 15 min, 4°C) were applied on both virus genera and the heat inactivation profiles were assessed. A significant decrease in the detected quantity of BoHV-1 DNA and ASFV DNA after heat-treatment (60°C and 95°C) and consequent incubation with Pd compounds was observed. BB-PdCl 2 and PdCl 2-COD could help to distinguish between infectious and non-infectious enveloped DNA viruses such as BoHV-1 or ASFV.

• Klíčová slova
• African swine fever virus, bovine herpesvirus-1, molecular methods, palladium compounds, viability PCR,
• MeSH
• biotest veterinární   MeSH
• bovinní herpesvirus 1 * MeSH
• DNA viry MeSH
• kvantitativní polymerázová řetězová reakce veterinární   MeSH
• palladium farmakologie   MeSH
• prasata MeSH
• virus afrického moru prasat * genetika   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• palladium MeSH

This paper concerns the formation of biofilm in bacteria of the genus Arcobacter. A multiplex polymerase chain reaction (PCR) method was introduced and optimized for detecting biofilm while using the intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA), first for analysis of strains of the genus Arcobacter from a collection, and then applied to samples of prepared biofilms. The results of the study indicate considerable variability among species of bacteria within the genus Arcobacter. The EMA-PMA PCR method can distinguish viable cells from dead cells and is therefore suitable for determining the viability of cells.

• MeSH
• azidy chemie   MeSH
• biofilmy * MeSH
• Campylobacter genetika   izolace a purifikace   fyziologie   MeSH
• interkalátory chemie   MeSH
• mikrobiální viabilita * MeSH
• multiplexová polymerázová řetězová reakce metody   MeSH
• propidium analogy a deriváty   chemie   MeSH
• Publikační typ
• časopisecké články MeSH
• hodnotící studie MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 8-azidoethidium MeSH Prohlížeč
• azidy MeSH
• interkalátory MeSH
• propidium monoazide MeSH Prohlížeč
• propidium MeSH

Quantitative PCR (qPCR) has become a frequently employed direct method for the detection and quantification of Mycobacterium avium subsp. paratuberculosis (MAP). The quantity of MAP determined by qPCR, however, may be affected by the type of qPCR quantification standard used (PCR product, plasmid, genomic DNA) and the way in which standard DNA quantity is determined (absorbance, fluorescence). In practice, this can be reflected in the inability to properly compare quantitative data from the same qPCR assays in different laboratories. Thus, the aim of this study was to prepare a prototype of an international MAP reference standard, which could be used to calibrate routinely used qPCR quantification standards in various laboratories to promote clinical data comparability. Considering stability, storage and shipment issues, a lyophilised fecal suspension artificially contaminated with a MAP reference strain was chosen as the most suitable form of the standard. The effect of five types of lyophilisation matrices on standard stability was monitored on 2-weeks interval basis for 4 months by F57 qPCR. The lyophilisation matrix with 10% skimmed milk provided the best recovery and stability in time and was thus selected for subsequent comparative testing of the standard involving six diagnostic and research laboratories, where DNA isolation and qPCR assay procedures were performed with the parallel use of the identical supplied genomic DNA solution. Furthermore, the effect of storage conditions on the standard stability was tested for at least 6 months. The storage at room temperature in the dark and under light, at + 4 °C, - 20 °C and - 80 °C showed no significant changes in the stability, and also no substantial changes in MAP viability were found using phage amplification assay. The prepared MAP quantification standard provided homogeneous and reproducible results demonstrating its suitability for utilisation as an international reference qPCR standard.

• MeSH
• DNA bakterií klasifikace   genetika   MeSH
• feces chemie   mikrobiologie   MeSH
• kvantitativní polymerázová řetězová reakce normy   MeSH
• lyofilizace MeSH
• Mycobacterium avium subsp. paratuberculosis klasifikace   genetika   izolace a purifikace   MeSH
• nemoci skotu diagnóza   MeSH
• paratuberkulóza diagnóza   mikrobiologie   MeSH
• referenční standardy MeSH
• senzitivita a specificita MeSH
• skot MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• DNA bakterií MeSH

Mycobacterium avium paratuberculosis (MAP), etiological agent of paratuberculosis in ruminants, is able to survive extreme conditions like very low pH (stomach), high temperature (pasteurization) or low temperature (refrigerated storage). Cheese, infant powder milk, cream and other milk and dairy products might thus be considered as possible sources of MAP for humans. The aim of this study was to investigate the survival of two MAP field isolates during fermentation of three different types of soured milk products (SMP; yogurt, acidophilus milk and kefir) under laboratory conditions. Pasteurized MAP-free milk was artificially contaminated with 10(6)MAPcells/mL and survival and absolute numbers of MAP were monitored during fermentation (4 or 16 h) and after six weeks of storage at 4°C by culture and quantitative real time PCR (qPCR). Viability of MAP was determined by culture using Herrold's egg yolk medium and Middlebrook 7H10 with antibiotics, supplemented with Mycobactin J and incubated at 37°C for up to 12 weeks. The absolute numbers of MAP were quantified by previously published qPCR assays targeting F57 and IS900 loci in MAP genome. We herein confirm that MAP can survive pH reduction, however, longer exposure to pH below 4 in SMP seems to be critical because it inhibits growth. Therefore, it is suggested that probiotic cultures that can decrease pH below 4 during fermentation could provide better inactivation of MAP in SMP.

• MeSH
• fermentace MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• mléčné výrobky mikrobiologie   MeSH
• mléko mikrobiologie   MeSH
• Mycobacterium avium subsp. paratuberculosis genetika   růst a vývoj   izolace a purifikace   MeSH
• paratuberkulóza mikrobiologie   MeSH
• pasterizace MeSH
• probiotika MeSH
• sýr MeSH
• vysoká teplota MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH