Recovery of Mycobacteria from Heavily Contaminated Environmental Matrices

For epidemiology studies, a decontamination method using a solution containing 4.0% NaOH and 0.5% tetradecyltrimethylammonium bromide (TDAB) represents a relatively simple and universal procedure for processing heavily microbially contaminated matrices together with increase of mycobacteria yield and elimination of gross contamination. A contamination rate only averaging 7.3% (2.4% in Cluster S; 6.9%% in Cluster R and 12.6%% in Cluster E) was found in 787 examined environmental samples. Mycobacteria were cultured from 28.5% of 274 soil and water sediments samples (Cluster S), 60.2% of 251 samples of raw and processed peat and other horticultural substrates (Cluster R), and 29.4% of 262 faecal samples along with other samples of animal origin (Cluster E). A total of 38 species of slow and rapidly growing mycobacteria were isolated. M. avium ssp. hominissuis, M. fortuitum, and M. malmoense were the species most often isolated. The parameters for the quantitative detection of mycobacteria by PCR can be significantly refined by treating the sample suspension before DNA isolation with PMA (propidium monoazide) solution. This effectively eliminates DNA residue from both dead mycobacterial cells and potentially interfering DNA segments present from other microbial flora. In terms of human exposure risk assessment, the potential exposure to live non-tuberculous mycobacteria can be more accurately determined.

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Rapid one-step enzyme immunoassay and lateral flow immunochromatographic assay for colistin in animal feed and food

Journal of Animal Science and Biotechnology ◽

10.1186/s40104-019-0389-7 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 5

Author(s):

Jiayi Wang ◽

Jinyu Zhou ◽

Yiqiang Chen ◽

Xinpei Zhang ◽

Yongpeng Jin ◽

...

Keyword(s):

Animal Feed ◽

Animal Production ◽

Lateral Flow ◽

Immunochromatographic Assay ◽

Animal Origin ◽

Quantitative Detection ◽

One Step ◽

Assay Time ◽

Animal Origin Food ◽

Lateral Flow Immunochromatographic Assay

Abstract Background Colistin (polymyxin E) is a kind of peptide antibiotic which has been approved in animal production for the purposes of disease prevention, treatment, and growth promotion. However, the wide use of colistin in animal feed may accelerate the spread of colistin-resistance gene MCR-1 from animal production to human beings, and its residue in animal-origin food may also pose serious health hazards to humans. Thus, it is necessary to develop corresponding analytical methods to monitor the addition of colistin in animal feed and the colistin residue in animal-origin food. Results A one-step enzyme-linked immunosorbent assay (ELISA) and a lateral flow immunochromatographic assay (LFIA) for colistin were developed based on a newly developed monoclonal antibody. The ELISA showed a 50% inhibition value (IC50) of 9.7 ng/mL with assay time less than 60 min, while the LFIA had a strip reader-based detection limit of 0.87 ng/mL in phosphate buffer with assay time less than 15 min. For reducing the non-specific adsorption of colistin onto sample vial, the components of sample extraction solution were optimized and proved to greatly improve the assay accuracy. The spiked recovery experiment showed that the recoveries of colistin from feed, milk and meat samples were in the range of 77.83% to 113.38% with coefficient of variations less than 13% by ELISA analysis and less than 18% by LFIA analysis, respectively. Furthermore, actual sample analysis indicated that the two immunoassays can produce results consistent with instrumental analysis. Conclusions The developed assays can be used for rapid qualitative or quantitative detection of colistin in animal feed and food.

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The Revised Method of Quantitative Detection of Animal-Origin Bovine and Porcine Gelatin Difference Using Surface Plasmon Resonance Based Biosensor

Materials Science Forum ◽

10.4028/www.scientific.net/msf.948.146 ◽

2019 ◽

Vol 948 ◽

pp. 146-152

Author(s):

Devy Pramudyah Wardani ◽

Muhammad Arifin ◽

Kamsul Abraha

Keyword(s):

Surface Plasmon Resonance ◽

Surface Plasmon ◽

Plasmon Resonance ◽

Sensor Response ◽

Animal Origin ◽

Quantitative Detection ◽

Response Models ◽

Gelatin Concentration ◽

The Difference ◽

Different Characteristics

We have previously reported the surface plasmon resonance (SPR)-based biosensor ability for quantitatively differentiating bovine and porcine gelatin has been done by us before. However, it has some inaccuracies. By improving the method of detection, the results of this study shows that the difference between bovine and porcine gelatin was more distinguishable. The sensor response models acquired were nonlinear as in the previous study. However, they show different characteristics. The sensitivities of the sensor obtained are higher than those of the previous ones, i.e., 3.04o and 4.29o for bovine and porcine gelatin concentration change of 0.1%, respectively. And the sensor’s LOD and LOQ towards both gelatin concentrations were 0.22% and 0.74% (w/w), respectively.

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Molecular Characterization and Antimicrobial Resistance Profile of Methicillin-Resistant Staphylococcus aureus in Retail Chicken

Journal of Food Protection ◽

10.4315/0362-028x.jfp-15-150 ◽

2015 ◽

Vol 78 (10) ◽

pp. 1879-1884 ◽

Cited By ~ 24

Author(s):

KHALID IBRAHIM SALLAM ◽

SAMIR MOHAMMED ABD-ELGHANY ◽

MOHAMED ELHADIDY ◽

TOMOHIRO TAMURA

Keyword(s):

Staphylococcus Aureus ◽

Antimicrobial Resistance ◽

Molecular Characterization ◽

Methicillin Resistant Staphylococcus Aureus ◽

Meca Gene ◽

Multidrug Resistant ◽

Animal Origin ◽

Contamination Rate ◽

Methicillin Resistant ◽

Food Of Animal Origin

The emergence of livestock-associated methicillin-resistant Staphylococcus aureus (MRSA) in food-producing animals is of increasing interest, raising questions about the presence of MRSA in food of animal origin and potential sources of transmission to humans via the food chain. In this study, the prevalence, molecular characterization, virulence factors, and antimicrobial susceptibility patterns of MRSA isolates from 200 retail raw chicken samples in Egypt were determined. MRSA was detected by positive amplification of the mecA gene in 38% (76 of 200) of chicken samples analyzed. This represents a potential public health threat in Egypt, as this contamination rate seems to be the highest among other studies reported worldwide. Furthermore, genes encoding α-hemolysin (hla) and staphylococcal enterotoxins (sea, seb, and sec) were detected in all of the 288 MRSA isolates. Nonetheless, none of the strains tested carried tst, the gene encoding toxic shock syndrome toxin 1. Antimicrobial resistance of MRSA isolates was most frequently detected against penicillin (93.4%), ampicillin (88.9%), and cloxacillin (83.3%). These results suggest that retail chicken might be a significant potential source for transmission of multidrug-resistant and toxigenic S. aureus in Egypt. This underlines the need for stricter hygienic measures in chicken production in Egypt to minimize the risk of transmission of these strains to consumers. To the best of our knowledge, this is the first study that reports the isolation and molecular characterization of MRSA in retail chicken samples in Egypt.

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Efficiency of DNA Isolation Methods Based on Silica Columns and Magnetic Separation Tested for the Detection of Mycobacterium avium Subsp. Paratuberculosis in Milk and Faeces

Materials ◽

10.3390/ma13225112 ◽

2020 ◽

Vol 13 (22) ◽

pp. 5112

Author(s):

Marketa Husakova ◽

Petr Kralik ◽

Vladimir Babak ◽

Iva Slana

Keyword(s):

Magnetic Separation ◽

Mycobacterium Avium ◽

Magnetic Beads ◽

Dna Isolation ◽

Mycobacterium Avium Subsp ◽

Effective Control ◽

Silica Column ◽

Faecal Samples ◽

Isolation Methods ◽

Mycobacterium Avium Subsp Paratuberculosis

Timely and reliable detection of animals shedding Mycobacterium avium subsp. paratuberculosis (MAP) should help to effectively identify infected animals and limit infection transmission at early stages to ensure effective control of paratuberculosis. The aim of the study was to compare DNA extraction methods and evaluate isolation efficiency using milk and faecal samples artificially contaminated by MAP with a focus on modern instrumental automatic DNA isolation procedures based on magnetic separation. In parallel, an automatic and manual version of magnetic separation and two methods of faecal samples preparation were compared. Commercially available DNA isolation kits were evaluated, and the selected kits were used in a trial of automatic magnetic beads-based isolation and compared with the manual version of each kit. Detection of the single copy element F57 was performed by qPCR to quantify MAP and determine the isolation efficiency. The evaluated kits showed significant differences in DNA isolation efficiencies. The best results were observed with the silica column Blood and Tissue kit for milk and Zymo Research for faeces. The highest isolation efficiency for magnetic separation was achieved with MagMAX for both matrices. The magnetic separation and silica column isolation methods used in this study represent frequently used methods in mycobacterial diagnostics.

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Comparison of culture-based, vital stain and PMA-qPCR methods for the quantitative detection of viable hookworm ova

Water Science & Technology ◽

10.2166/wst.2017.142 ◽

2017 ◽

Vol 75 (11) ◽

pp. 2615-2621 ◽

Cited By ~ 4

Author(s):

P. Gyawali ◽

J. P. S. Sidhu ◽

W. Ahmed ◽

P. Jagals ◽

S. Toze

Keyword(s):

Environmental Samples ◽

Quantitative Measurement ◽

Quantitative Polymerase Chain Reaction ◽

Detection Methods ◽

Quantitative Detection ◽

Propidium Monoazide ◽

Chain Reaction ◽

Vital Stain ◽

Polymerase Chain ◽

Hookworm Ova

Accurate quantitative measurement of viable hookworm ova from environmental samples is the key to controlling hookworm re-infections in the endemic regions. In this study, the accuracy of three quantitative detection methods [culture-based, vital stain and propidium monoazide-quantitative polymerase chain reaction (PMA-qPCR)] was evaluated by enumerating 1,000 ± 50 Ancylostoma caninum ova in the laboratory. The culture-based method was able to quantify an average of 397 ± 59 viable hookworm ova. Similarly, vital stain and PMA-qPCR methods quantified 644 ± 87 and 587 ± 91 viable ova, respectively. The numbers of viable ova estimated by the culture-based method were significantly (P < 0.05) lower than vital stain and PMA-qPCR methods. Therefore, both PMA-qPCR and vital stain methods appear to be suitable for the quantitative detection of viable hookworm ova. However, PMA-qPCR would be preferable over the vital stain method in scenarios where ova speciation is needed.

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Quantification of Live ‘Candidatus Liberibacter asiaticus’ Populations Using Real-Time PCR and Propidium Monoazide

Plant Disease ◽

10.1094/pdis-09-12-0880-re ◽

2013 ◽

Vol 97 (9) ◽

pp. 1158-1167 ◽

Cited By ~ 13

Author(s):

H. Hu ◽

M. J. Davis ◽

R. H. Brlansky

Keyword(s):

Real Time ◽

Propidium Monoazide ◽

Candidatus Liberibacter Asiaticus ◽

Disease Epidemiology ◽

Standard Curve ◽

Crucial Component ◽

Candidatus Liberibacter ◽

Greenhouse Plant ◽

Liberibacter Asiaticus ◽

Epidemiology Studies

Huanglongbing (HLB) is a devastating citrus disease. It is associated with a phloem-restricted bacterium, ‘Candidatus Liberibacter asiaticus’, and primarily transmitted by Asian citrus psyllid in Florida. Because Liberibacter cannot be cultured, early diagnosis of HLB relies on DNA-based polymerase chain reaction (PCR), including real-time quantitative (q)PCR. Although estimating genomes from live bacteria (GLB) is critical for HLB research, PCR does not distinguish between live and dead cells and, thus, does not estimate GLB in hosts. Propidium monoazide (PMA), a novel DNA-binding dye, has been successfully used on many bacterial pathogens to effectively remove DNA from dead cells but there is no report of its use on uncultured bacteria. In this study, PMA-qPCR protocols were first optimized to work with plant and psyllid samples, respectively. Both TissueLyser treatment and plant tissue were demonstrated to have an insignificant impact on the GLB detected by PMA-qPCR. Finally, a standard curve for GLB determination was successfully established between PMA-qPCR results and microscopic counts and then applied in two studies with different greenhouse plant samples. This rapid qPCR method provides a more accurate way to determine GLB in HLB hosts which, in turn, should benefit disease epidemiology studies and serve as a crucial component in HLB management.

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Detection of Echinococcus multilocularis in faeces by nested PCR with the use of diluted DNA samples

Polish Journal of Veterinary Sciences ◽

10.2478/pjvs-2014-0010 ◽

2014 ◽

Vol 17 (1) ◽

pp. 79-83 ◽

Cited By ~ 4

Author(s):

J. Karamon

Keyword(s):

Echinococcus Multilocularis ◽

Nested Pcr ◽

Dna Isolation ◽

False Negative ◽

Standard Samples ◽

Red Foxes ◽

Negative Results ◽

False Negative Results ◽

Faecal Samples ◽

Positive Results

AbstractThe aim of this study was to choose the optimal variant of PCR examination of faeces to detect Echinococcus multilocularis infection which would allow to reduce the influence of different inhibitors in faeces. The investigation was carried out by comparison of 3 different methods of DNA isolation from faeces and different DNA dilutions used in PCR. Thirty five intestines of red foxes were used. Small intestines were examined by the sedimentation and counting technique (SCT). Faeces were collected from the rectum for PCR and flotation. DNA were isolated with the use of 3 different methods. Two methods were dedicated for faeces: method 1 (M1) - for larger samples and method 2 (M2) - for standard samples. The third method, method 3 (M3), was not dedicated for faeces. DNA samples were tested by nested PCR in 6 variants: not diluted (1/1) and 5 diluted (1/2.5, 1/5, 1/10. 1/20, 1/40). E. multilocularis was found by SCT in 18 from 35 (51.4%) intestines. Taenia-type eggs were detected only in 20.0% of faecal samples. In PCR the highest number of positive results (45.7%) were obtained during examination of DNA isolated by M1 method, and then 40.0% and 34.3%, respectively, for M2 and M3. In some samples positive results in PCR were obtained only in diluted DNA. For example, 8 from 12 positive samples isolated by M3 method gave the PCR negative results in non-diluted DNA and positive only after dilution 1:2.5, 1:10 or 1:20. Also 3 samples isolated by methods dedicated for stool gave positive results only after DNA dilution. The investigation has revealed that in copro-PCR for detection of E. multilocularis infection additional using of diluted DNA (besides non diluted) can avoid false negative results causing by PCR inhibition. In the best method of DNA isolation (M1), the use of non diluted DNA sample together with diluted in proportion 1:10 seems to be optimal.

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Rapid Quantification of Viable Campylobacter Bacteria on Chicken Carcasses, Using Real-Time PCR and Propidium Monoazide Treatment, as a Tool for Quantitative Risk Assessment

Applied and Environmental Microbiology ◽

10.1128/aem.00411-10 ◽

2010 ◽

Vol 76 (15) ◽

pp. 5097-5104 ◽

Cited By ~ 111

Author(s):

M. H. Josefsen ◽

C. LÃ¯Â¿Â½fstrÃ¯Â¿Â½m ◽

T. B. Hansen ◽

L. S. Christensen ◽

J. E. Olsen ◽

...

Keyword(s):

Risk Assessment ◽

Real Time ◽

Control Strategies ◽

Quantitative Risk Assessment ◽

Quantitative Detection ◽

Amplification Efficiency ◽

Sample Treatment ◽

Propidium Monoazide ◽

Real Time Quantitative Pcr ◽

Chicken Carcass

ABSTRACT A number of intervention strategies against Campylobacter-contaminated poultry focus on postslaughter reduction of the number of cells, emphasizing the need for rapid and reliable quantitative detection of only viable Campylobacter bacteria. We present a new and rapid quantitative approach to the enumeration of food-borne Campylobacter bacteria that combines real-time quantitative PCR (Q-PCR) with simple propidium monoazide (PMA) sample treatment. In less than 3 h, this method generates a signal from only viable and viable but nonculturable (VBNC) Campylobacter bacteria with an intact membrane. The method's performance was evaluated by assessing the contributions to variability by individual chicken carcass rinse matrices, species of Campylobacter, and differences in efficiency of DNA extraction with differing cell inputs. The method was compared with culture-based enumeration on 50 naturally infected chickens. The cell contents correlated with cycle threshold (CT ) values (R 2 = 0.993), with a quantification range of 1 Ã¯Â¿Â½ 102 to 1 Ã¯Â¿Â½ 107 CFU/ml. The correlation between the Campylobacter counts obtained by PMA-PCR and culture on naturally contaminated chickens was high (R 2 = 0.844). The amplification efficiency of the Q-PCR method was not affected by the chicken rinse matrix or by the species of Campylobacter. No Q-PCR signals were obtained from artificially inoculated chicken rinse when PMA sample treatment was applied. In conclusion, this study presents a rapid tool for producing reliable quantitative data on viable Campylobacter bacteria in chicken carcass rinse. The proposed method does not detect DNA from dead Campylobacter bacteria but recognizes the infectious potential of the VBNC state and is thereby able to assess the effect of control strategies and provide trustworthy data for risk assessment.

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Quantitative Detection of Viable Bifidobacterium bifidum BF-1 Cells in Human Feces by Using Propidium Monoazide and Strain-Specific Primers

Applied and Environmental Microbiology ◽

10.1128/aem.03294-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2182-2188 ◽

Cited By ~ 28

Author(s):

Junji Fujimoto ◽

Koichi Watanabe

Keyword(s):

Dna Analysis ◽

Fermented Milk ◽

Bifidobacterium Bifidum ◽

Quantitative Detection ◽

Milk Product ◽

Propidium Monoazide ◽

Human Feces ◽

Fecal Samples ◽

Content Type ◽

Wet Weight

ABSTRACTWe developed a PCR-based method to detect and quantify viableBifidobacterium bifidumBF-1 cells in human feces. This method (PMA-qPCR) uses propidium monoazide (PMA) to distinguish viable from dead cells and quantitative PCR using a BF-1-specific primer set designed from the results of randomly amplified polymorphic DNA analysis. During long-term culture (10 days), the number of viable BF-1 cells detected by counting the number of CFU on modified MRS agar, by measuring the ATP contents converted to CFU, and by using PMA-qPCR decreased from about 1010to 106cells/ml; in contrast, the total number of (viable and dead) BF-1 cells detected by counting 4′,6-diamidino-2-phenylindolee (DAPI)-stained cells and by using qPCR without PMA and reverse transcription-qPCR remained constant. The number of viable BF-1 cells in fecal samples detected by using PMA-qPCR was highly and significantly correlated with the number of viable BF-1 cells added to the fecal samples, within the range of 105.3to 1010.3cells/g feces (wet weight) (r> 0.99,P< 0.001). After 12 healthy subjects ingested 1010.3to 1011.0CFU of BF-1 in a fermented milk product daily for 28 days, 104.5 ± 1.5(mean ± standard deviation [SD]) BF-1 CFU/g was detected in fecal samples by using strain-specific selective agar; in contrast, 106.2 ± 0.4viable BF-1 cells/g were detected by using PMA-qPCR, and a total of 107.6 ± 0.7BF-1 cells/g were detected by using qPCR without PMA. Thus, the number of viable BF-1 cells detected by PMA-qPCR was about 50 times higher (P< 0.01) than that detected by the culture-dependent method. We conclude that strain-specific PMA-qPCR can be used to quickly and accurately evaluate viable BF-1 in feces.

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