ABSTRACT This study compared colony counts of mixed bacterial cultures on chromogenic spread plates using manual and Color QCount (Spiral Biotech, Inc., Norwood, MA) automated methods. Inoculum levels spanned 30–300 cfu/mL, 26 agar types were used and 581 plates were analyzed. Plates were prepared according to manufacturers' instructions, manually counted once by two scientists and counted in duplicate automatically. The correlation coefficient comparing automated and manual counts for the pooled population of data was 0.987. The slope and intercept for the linear regression line were 1.0067 and 0.031, respectively. The mean log value difference between automated and manual counts for pooled data was−0.042. The mean log value differences between manual and automated counts demonstrated that 83.8% of plates analyzed were within 0.1 log, and 98.2% were within 0.2 log. These results demonstrate that the Color QCount automatic counter is a suitable alternative to the standard method of manually counting colored colonies on chromogenic agar plates.PRACTICAL APPLICATIONSA variety of microbiology applications require users to count bacterial colonies on agar plates. Colonies are counted to 1) assess levels of contamination in the environment, water, food and dairy products; 2) conduct preservative and efficacy studies; and 3) determine the quality of pharmaceutical and medical products with microbial limits testing. There are several sources of variability in plating including inoculating errors, sampling errors, incubation errors, and counting errors. Errors in counting may worsen with user fatigue.Using an automated colony counter to complete the tedious job of manually counting bacterial colonies on agar plates will not only improve efficiency in microbiology laboratories by saving valuable time for alternative tasks but also may improve the accuracy and consistency of the counts.
ABSTRACT In this study, we demonstrated the simultaneous detection of Escherichia coli and Salmonella enteritidis, by coupling immunomagnetic separation (IMS) with quantum dots (QDs) labeling. QDs having different emission wavelengths were conjugated with anti‐ E. coli and anti‐ Salmonella antibodies. QD–antibody conjugates were used to label immunomagnetically separated bacteria and the fluorescence intensities were measured for enumerations of both species. The concentrations of primary antibodies used in IMS, the ratio of QDs to antibodies during the conjugation and the concentration of QD–antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead–bacteria complex and QDs was eliminated by separating QDs from the complex using sodium dodecyl sulfate solution. The fluorescence intensities due to the capturing of different concentrations of bacteria were measured and the working ranges were found to be 5 × 10 2 to 5 × 10 5 cfu/mL for E. coli and 4 × 10 2 to 4 × 10 5 cfu/mL for S. enteritidis . PRACTICAL APPLICATIONS In this study, antibody‐conjugated multicolor quantum dots (QDs) were used for simultaneous detection of Escherichia coli and Salmonella enteritidis . The results of this study indicate that QD labels can be used in multiplex, rapid and selective detection of bacteria with detection limits comparable with those of many novel methods in cases where the assay conditions are optimized. Furthermore, the assay can be modified for the simultaneous detection of more than two species through using QD labels having different emission wavelengths.
ABSTRACT Despite advances in the specificity and sensitivity of molecular biological technologies, the efficient recovery of DNA from low‐biomass samples remains extremely challenging. Optimal methods to purify biomolecules from such environments should (1) achieve the greatest total yield and (2) reflect the true microbial diversity of the sample. These attributes were assessed from five DNA purification regimes: a standard‐manual procedure, MoBio Ultraclean and Promega Wizard kits, and an automated Axcyte AutoLyser method with and without bead‐beating agitation. A homogenous mixture of known concentrations of nine distinct bacterial lineages isolated from low‐biomass environments was prepared and suitable aliquots of subsamples were processed in parallel. DNA products from each of these methods were then subjected to polymerase chain reaction (PCR), quantitative PCR and 16S rRNA clone‐library analysis. The Axcyte AutoLyser outperformed all other purification regimes examined. This automated method consistently both yielded the highest concentration of PCR‐amplifiable DNA, and reported species composition most consistent with the starting solution. PRACTICAL APPLICATIONS This communication carefully examines the effectiveness of common DNA purification regimes as well as an automated method. Comparative analyses convincingly demonstrate that the different methods not only result in different recovery of genomic DNA, but more importantly, different estimations of microbial diversity in the sample. This report will hopefully inspire investigators from various industries (pharmaceutical, ecological, medical, semiconductor, etc.) who find themselves in the initial phases of large‐scale studies to devote a significant effort into optimizing sample extraction protocols to achieve the most accurate information.
ABSTRACT The aim of the present research was to isolate and identify cellulolytic bacteria from the gut of the local termite Coptotermes curvignathus (Holmgren) present in the vicinity of the University of Putra Malaysia. The isolates were cultured in a medium containing carboxymethyl‐cellulose and cellobiose. The bacterial species were tentatively identified by using the Biolog reader as well as the Bergey's manual and later confirmed by 16S rRNA sequence homology. The species were all novel strains and identified as Bacillus cereus strain Razmin A, Enterobacter aerogenes strain Razmin B, Enterobacter cloacae strain Razmin C, Chryseobacterium kwangyangense strain Cb and Acinetobacter strain Raminalimon. Biolog reader was not able to identify one of the bacterial species in which it was identified as C. kwangyangense strain Cb by 16S rRNA sequence homology. The GenBank NCBI/EMBL accession numbers for the bacterial strains are EU294508, EU305608, EU305609, EU169201 and EU332791 for B. cereus Razmin A, E. aerogenes strain Razmin B, E. cloacae strain Razmin C, C. kwangyangense strain Cb and Acinetobacter strain Raminalimon, respectively.PRACTICAL APPLICATIONSThe experiment describes the isolation and identification of cellulolytic bacteria from termites, using Biolog reader and polymerase chain reaction technique. Termites are common insects in tropical regions and thrive on wood and cellulolytic materials. Hence, the bacteria isolated may be useful in the degradation of cellulosic materials to increase their digestibility and possibly production of metabolites and enzymes.
ABSTRACT Aceticlastic and hydrogenotrophic methanogens were comparatively investigated by 16S rRNA‐based fluorescence in situ hybridization (FISH) technique combined with Confocal Scanning Laser Microscopy (CSLM) to evaluate temperature influence on two functional groups of methanogens. Microorganisms were grown in the form of anaerobic granular sludge in two lab‐scale Upflow Anaerobic Sludge Bed (UASB) reactors fed with synthetic wastewater and operated at psychrophilic and mesophilic temperature conditions. Since hydrogenotrophic and aceticlastic methanogenesis are known to play a crucial role, digestion may be effectively blocked if these processes are ceased due to unfavorable environmental temperature conditions. Mesophilic (35 ± 2C) and psychrophilic (10 ± 1C) reactors were inoculated by the same mesophilic granules taken from a full‐scale UASB reactor. According to FISH results, Archaea representing methanogens were found dominating at bottom sampling ports of reactors and dominant member of Archaea was the genus aceticlastic Methanosaeta (MX825). Other aceticlastic methanogen such as Methanosarcina‐like (SARCI645) species were also identified which were higher in psychrophilic granules. Members of order Methanobacteriales (MB310) constituted the major hydrogenothrophic methanogens in both reactors, whereas other hydrogenothrophic methanogens members of order Methanococcales (MC1109) and Methanogenium (MG1200) relatives were absent. Abundance of originally mesophilic Methanosaeta‐related Archaea under low temperature conditions demonstrated the adaptive capacity of microorganisms to psychrophilic conditions. This might be attributed to enzymatic alterations in Methanosaeta cells originating from seed sludge, which were exposed to sub‐mesophilic temperatures at start‐up and then to psychrophilic conditions during gradual temperature decreases. Both sludge granules remained undisturbed and kept their rigidity even after exposure to very low temperatures. Quantification study was done using Image Pro‐plus Version 4.0. Archaeal cells detected with probe ARC915 were found around 60% of the 4,6‐diamidino‐2‐phenylindole stained cells at both reactors. Methanosaeta spp./Archaea ratios for mesophilic and psychrophilic UASB reactors were calculated as 71% and 84%, respectively. FISH and CSLM results together with quantification study showed that the microbial community of anaerobic granular sludges was dominated by Methanosaeta spp. which was identified as the major methanogenic Archaea.PRACTICAL APPLICATIONSSince effects on bioreactor performances were correlated to variations in microbial diversity, investigation of methanogenic diversity in anaerobic reactors is important during operation. Identification of microorganisms should be done for formation, stability, and physiological properties of the biomass in bioreactors, which is a key factor during process performance evaluation. Monitoring the changes in dominant archea of seed added at start‐up, gives information on system performance. A detailed investigation on diversity, structure and function of mixed microbial communities in anaerobic reactors is necessary to improve treatment efficiency and stability against inhibitory compounds. Among 16S rRNA based microbial identification techniques, FISH is a well‐known technique that helps determination of dominant species by giving a detailed analysis of microbial populations while CSLM is the most appropriate method for monitoring the formation of 3‐D structure of microbial biomass. In this study, temperature influence on aceticlastic and hydrogenotrophic methanogen groups was evaluated using FISH coupled with CSLM.
ABSTRACT A loop‐mediated isothermal amplification (LAMP)‐based assay for the detection of Vibrio anguillarum O2β, the causative agent of vibriosis in Atlantic cod, Gadus morhua , was developed. Five sets of primers targeting the flanking regions of the genes, hemolysin and amiB , which encodes the peptidoglycan hydrolase N‐acetylmuramoyl‐L‐alanine amidase of the pathogen were designed. The primers were specific for the detection of Vibrio anguillarum O2β with no cross reactions to other bacterial pathogens commonly infecting Atlantic cod, e.g., Yersinia ruckeri, Francisella piscicida, Aeromonas salmonicida and some endogenous bacteria found in the gut of Atlantic cod. The detection limit of the assay was 10 pg of bacterial DNA/mL or 10 fg of bacterial DNA per LAMP reaction; however, the sensitivity of the reaction decreased by 3‐log dilution in the presence of 1 mg/mL of mucus samples as inhibitors. Nevertheless, the assay can be potentially used as a direct and nondestructive method to detect the pathogen in the fish, thus making the assay less time‐consuming. These results suggest that the LAMP assay is a specific and sensitive molecular method to detect vibriosis in Atlantic cod and will be helpful for routine surveillance programs in aquaculture systems. PRACTICAL APPLICATIONS Loop‐mediated isothermal amplification (LAMP), a potential diagnostic technique performed under isothermal conditions without the use of any sophisticated equipment, can be used for the rapid and early detection of Vibrio anguillarum 02β causing vibriosis in Atlantic cod. The developed assay is highly sensitive and can be used to detect the pathogen prior to the onset of infection, thus, immediate measures can be applied to prevent heavy losses of the cultured stock.
ABSTRACT The spread‐plate and double agar layer (DAL) methods are common for the enumeration of bacteria and viral indicators (bacteriophages). However, they may become cumbersome in large matrix experiments or when the titer of the organism varies by several orders of magnitude. A bacterial spot‐titer assay has been available for decades but has not been adapted to bacteriophages and has rarely been applied to the analysis of environmental samples. In this study, a spot‐titer culture‐based method was investigated for bacteria and bacteriophages. The method involves spot‐plating replicate 10‐µl volumes of several sample dilutions on a single plate, incubating, and counting colonies or plaques. Parallel assays of laboratory cultures and environmentally isolated organisms show that the spot‐titer method is equally straightforward and statistically comparable to the spread‐plate and DAL methods ( R 2 = 0.989 for laboratory strains and R 2 = 0.972 for environmental samples), while more cost‐ and labor‐efficient. PRACTICAL APPLICATIONS The spread‐plate and double agar layer (DAL) methods currently used for enumeration of bacteria and viral indicators, may become labor‐ and resource‐intensive (culture media, plates, technician time and incubator space) in large matrix experiments, which are often needed in the laboratory to evaluate environmental conditions. The spot‐titer method has several advantages over the spread‐plate and DAL methods: (1) it requires less time to dispense spots than to spread the microbe; (2) it uses fewer materials (15–20% of the laboratory supplies as the traditional methods); (3) it requires less effort; and (4) since the sample is distributed in distinct spots, colony/plaque counting is faster and less labor intensive. The spot‐titer method was found to economize resources without sacrificing accuracy or precision, and is a practical method for routine use in large matrix experiments (e.g., survival or disinfection studies) and enumeration of high‐titer environmental samples.
ABSTRACT The aim of our work was to develop a new molecular method for the simultaneous detection of Salmonella spp., Listeria monocytogenes and Escherichia coli O157 directly in milk samples. Three specific target sequences were chosen for a multiplex polymerase chain reaction (PCR) assay: a 155‐bp region of the Salmonella spp. tetrathionate reductase ( ttr ) locus; a 173‐bp region of the L. monocytogenes listeriolysin O gene ( hlyA ); a 217‐bp region of the E. coli O157 lipopolysaccharide gene ( rfbE ). An internal amplification control was also included to detect PCR inhibition. In addition, a magnetic‐based extraction method was also developed to isolate PCR‐ready DNA from milk. The assay was able to detect, whether alone or mixed, also with a difference of 2 log units, as few as 10 2 cells of each pathogen per 10 mL of spiked milk. PRACTICAL APPLICATIONS Escherichia coli O157, Salmonella spp. and Listeria monocytogenes are among the most dangerous foodborne bacterial pathogens in terms of human health and disease, and their detection through current microbiological culture methods involves considerable spending of time and effort. Therefore, the development of rapid and sensitive diagnostic methods is needed. The magnetic DNA extraction proposed in this study concentrates the target organism DNA, removes the inhibitors and presents the additional advantages of being safe and suitable for automation systems. Moreover, the developed multiplex polymerase chain reaction (mPCR) enables the simultaneous detection of more than one target sequence in the same reaction, producing considerable cost and reagent savings. The assay, combining magnetic extraction and mPCR, has proven to be a valuable and cost‐effective tool for monitoring the presence of these pathogens directly in milk samples. Therefore, it could provide a major contribution to efficiency and logistics of molecular diagnostics.
ABSTRACT A loop‐mediated isothermal amplification (LAMP)‐based assay was developed for the detection of atypical furunculosis, caused by Aeromonas salmonicida in Atlantic cod, Gadus morhua. Gene gyrB encoding the B subunit of DNA gyrase present in the pathogen was selected for designing five sets of primers targeting the flanking regions of the gene. The primers were specific for the detection of A. salmonicida with no cross reactions to other bacterial pathogens commonly infecting Atlantic cod, e.g., Vibrio anguillarum, Francisella piscicida, Yersinia ruckeri and some endogenous bacteria found in the gut of Atlantic cod. The detection limit of the assay was 1 picogram of bacterial DNA mL−1, whereas there was a decrease in detection limit by 1 log dilution in the presence of mucus as inhibitor. Because of its specificity and sensitivity, LAMP can be considered a useful tool in routine surveillance programs in aquaculture systems for monitoring atypical furunculosis in Atlantic cod and other marine species.PRACTICAL APPLICATIONSLAMP is a potential diagnostic technique that can be used for the rapid and early detection of atypical Aeromonas salmonicida, the causative agent of furunculosis in Atlantic cod. This technique does not require sophisticated equipment and can be performed under isothermal conditions. The assay is highly sensitive to enable detection of the pathogen prior to the onset of infection, thus, mitigating measures can be applied to prevent heavy losses of the cultured stock.
ABSTRACT A microplate assay was modified for the detection of antimicrobial activity in plant extracts. The aim was to develop an in vitro assay that could rapidly screen plant extracts to provide quantitative data on inhibition of microbial growth. A spectrophotometric assay using a microplate with serial dilutions of the plant extract and the bacteria was developed. Two bacteria, Staphylococcus aureus and Escherichia coli, were used for this study. Essential oils, oregano (Origanum vulgare) and lemon myrtle (Backhousia citriodora), and three active components carvacrol, thymol and citral were evaluated. The reproducibility of the assay was high, with correlation coefficients (r2) of the replicates of lemon myrtle and oregano with S. aureus and E. coli between 0.9321 and 0.9816. Similarly, r2 values for carvacrol, thymol and citral were between 0.8455 and 0.9814. This assay could also be used to measure antimicrobial activity in plant extracts which vary in pH and color.PRACTICAL APPLICATIONSThis research could be used for the quantitative determination of antimicrobial activity in plant extracts. As inhibition of growth is expressed as a percentage from 0 to 100, this method could also be used to study synergies within plant extracts to enhance antimicrobial activity for future studies. When plant extracts are used in food as natural preservatives, the challenge is to use a concentration that does not interfere with the flavor of the food product. In general, the amount of plant extract required to extend the storage life of food is greater than that required to produce an acceptable flavor. The advantage of knowing the percent inhibition relates to the effect of synergies where the inhibitions less than 100% can be enhanced with other antimicrobial plant extracts, thereby reducing the amount of plant extract required.
ABSTRACT Three latex agglutination kits for rapid identification of Staphylococcus aureus were compared by testing a selection of isolates, which included methicillin resistant S. aureus, methicillin sensitive S. aureus and a diverse selection of coagulase‐negative staphylococci. The sensitivities of Monostaph Plus (Bionor Laboratories, Skien, Norway), Pastorex Plus (Bio‐Rad Laboratories, Hercules, CA) and Staphaurex Plus (Oxoid, Cambridge, UK) were 98.5, 98.3 and 98.3% respectively, and the specificities were 97.5, 97.0 and 96.5%, respectively. None of the kits detected Staphylococcus lugdunensis correctly. This evaluation shows that the Monostaph Plus agglutination kit performs well and comparable to Pastorex Staph‐Plus and Staphaurex Plus.PRACTICAL APPLICATIONSLatex agglutination kits are very useful in the routine identification of Staphylococcus aureus, but can occasionally give false‐positive or false‐negative results. Knowledge of how the various available kits are performing is, therefore, very important when selecting a kit for use. Correct identification of S. aureus is important, especially in relation to methicillin‐resistant S. aureus, since other staphylococci species are known to carry the methicillin‐resistance determinant mecA, but are of less clinical importance. This study is the first study comparing an improved version of the Monstaph‐Plus kit with other latex agglutination kits, and provides new data about this kit.
ABSTRACT Nested automated ribosomal intergenic spacer analysis (ARISA) was used to examine the community structure of epilithic biofilms in freshwater streams experiencing different levels of human impact. This molecular fingerprinting technique generated reproducible profiles of bacterial community structure that varied significantly between stream sites. Nested ARISA was determined to be a cost‐effective, high‐throughput approach to assess bacterial community composition from very small sample volumes, requiring little sampling effort and without the need for taxonomic identification of individual organisms. In combination with multidimensional scaling, nested ARISA provides a rapid and sensitive method to carry out complex analyses of bacterial community structure. PRACTICAL APPLICATIONS Nested automated ribosomal intergenic spacer analysis (ARISA) provides a high‐throughput molecular method with which to screen large numbers of environmental samples for differences in microbial community structure. This sensitive approach benefits assessments from small sample volumes or environments exhibiting reduced microbial biomass (both aquatic and terrestrial). Differences in bacterial community structure (obtained from ARISA profiles) could be used to characterize the impact of anthropogenic disturbance on freshwater systems, analogous to the current use of macroinvertebrate indicators of freshwater ecological health.
ABSTRACT The Fung Double Tube (FDT) and the newly developed CP AnaSelect Oxyplates methods can enumerate concentrations of Clostridium perfringens without need for external anaerobic generating systems. Because Clostridium perfringens is a reliable indicator of fecal contamination and it is one of the fastest growing fecal bacteria, these two methods were evaluated as feasible methods to screen recreational waters for sewage contamination. To increase the sensitivity and selectivity of these methods, three modifications were evaluated. The first modification was to pretreat the water samples using a microwave oven to attain high temperature (70C) for a short time (2.5 min) to reduce the interfering growth of non‐C. perfringens colonies on the selective media. The second modification was the addition of phosphatase reaction, thus enumerated colonies could be confirmed as C. perfringens. The third modification was to increase the sample volume for the FDT test from 5 to 10 mL/tube. The data collected showed that these modifications improved the selectivity and sensitivity of these two methods to enumerate C. perfringens from sewage‐contaminated water samples as well as environmental water samples such as streams, harbors, canals and coastal swimming beaches. The recovery efficiencies of the FDT and experimental CP AnaSelect Oxyplate for C. perfringens were similar to traditionally used membrane C. perfringens, tryptose sulfite cycloserine and Shahidi Ferguson perfringens agar media by membrane filtration technology, followed by incubation in anaerobic chambers. These results show that the modified FDT and CP AnaSelect Oxyplate methods are feasible and reliable methods to monitor environmental waters for C. perfringens. The FDT method is especially promising because it is simple, inexpensive and can produce results in 5–6 h.PRACTICAL APPLICATIONS Clostridium perfringens is currently being evaluated as a reliable indicator of sewage contamination in recreational waters. However, traditional methods to assay for C. perfringens are expensive and cumbersome because of the need for external anaerobic generating systems. In addition, target colonies must be confirmed by a second test. In this study, two methods are described, with capabilities to self‐generate anaerobic conditions and to immediately confirm the target colonies as C. perfringens. Moreover, one of the methods meets the difficult criterion of obtaining results in 6 h so decision on closing the beach can be reached on the same day the beach water sample is tested. Since these two methods are feasible and reliable, it will encourage many laboratories to assay their recreational waters for C. perfringens and a national database to determine the quality of recreational waters based on concentrations of C. perfringens can be developed.
ABSTRACT A new method was developed to rapidly monitor the Pseudomonas aeruginosa viable counts using alamar blue (AB). The 96‐well microtiter plates were used to perform the assay. This procedure is based on fluorogenic measurement as a result of reduction of nonfluorescent AB to red fluorescent form by the viable cells of P. aeruginosa. The correlation between conventional plate count and fluorogenic AB method was highly satisfactory for quantification of planktonic ( R 2 = 0.9487) and biofilm cells of P. aeruginosa ( R 2 = 0.9296). PRACTICAL APPLICATIONS The new fluorogenic method can rapidly monitor Pseudomonas aeruginosa counts in vitro with a high correlation with the conventional plating method. The results indicate that fluorogenic method requires much shorter time (2 h) than the conventional plate count (24 h), is a more cost‐effective way, quite amenable to high throughput, and continuous monitoring of P. aeruginosa viability is achievable in the kinetic in vitro models without interference with the cell viability.
ABSTRACT Two multiplex polymerase chain reaction (mPCR) assays were developed for simultaneous detection of antimicrobial resistance genes in Escherichia coli and enterococci. Oligonucleotide primer sets were designed and formulated. Primer set I was used to detect resistance genes including tetracycline (tetM and tetA), chloramphenicol (cat1 and cmlA) and sulfonamide (sul1), in 20 E. coli isolates. Primer set II was designed to identify three target genes including tetM, tetL and ermB of tetracycline and erythromycin resistance genes, respectively. In the final volume of 25 µL, optimal concentrations of mPCR primer for investigation of E. coli and enterococci resistance genes were 0.1, 0.4, 0.2, 0.4 and 0.2 µM of tetA, tetM, cat1, cmlA and sul1, and 0.2, 0.2 and 0.1 µM of tetM, tetL and ermB, respectively. The optimal annealing temperatures were 51 and 57C to detect all expected resistance genes of E. coli and enterococci, respectively. The amplicon sizes ranged from 171 to 847 bp for E. coli and 171 to 505 bp for enterococci, differing by at least 83 bp to simplify gel electrophoretic separation. The correlation between dot blot hybridization and mPCR results were investigated. Two of the 20 E. coli isolates showed positive results by mPCR and negative results by hybridization assay. The same results were observed with two different methods among 20 enterococci isolates indicating that the targets were amplified efficiently. The developed mPCR assay is a simple, rapid and useful method for genotyping detection of multiple resistance genes in single reaction.PRACTICAL APPLICATIONSMultiplex polymerase chain reaction (mPCR) can allow the detection of various antimicrobial resistance genes in one PCR reaction tube. The technique is fast and reliable for rapid screening of multiple resistance genes. In this study, we focused on the simultaneous detection of five and three most commonly found resistance genes in Escherichia coli and enterococci isolates, respectively. Our research has shown that the mPCR method could be expanded to include the determination of other antibiotic resistance genes of interest. The system described here can decrease PCR reagent cost by multiple resistance genes detection in each reaction tube. The high throughput and cost‐effective mPCR system developed in this study could provide a powerful tool for more accurate detection of various antimicrobial resistance genes associated with E. coli and enterococci isolates.
ABSTRACT There is no rapid diagnostic technique at medical examiners' offices to determine if a decedent is infected with Mycobacterium tuberculosis. Present diagnostic testing requires at least 1 month for results. The RAPTOR, a portable, automated fiber optic evanescent wave biosensor, was used as the platform to develop more rapid assays to detect M. tuberculosis from lung tissue. Positive biosensor detection was obtained 80% of the time at cell concentrations of 106 cells/mL, 96% of the time at 107 cells/mL, and 99% of the time at 108 cells/mL of live attenuated M. tuberculosis (ATCC 25177) suspended in phosphate‐buffered saline with 0.1% Tween 20 (PBST). Live attenuated M. tuberculosis suspended in PBST and seeded into decedent lung tissue was tested using the RAPTOR. Positive detection was obtained 96% of the time at 107 cells/mL and 100% of the time at 108 cells/mL. Detection of M. tuberculosis in lung tissue homogenate was possible within 3 h.PRACTICAL APPLICATIONSDevelopment of a rapid method to detect Mycobacterium tuberculosis from lung tissue at autopsy would be a significant benefit to medical examiners' offices and pathologists worldwide. Rapid diagnosis would enable those possibly exposed to an infected decedent to be informed of M. tuberculosis infection in a timely manner. Biosensor assays on seeded lung tissue show it is possible to detect M. tuberculosis in 3 h using the RAPTOR biosensor, instead of 1 month using current, conventional tests, when there are 107 or more cells in the assay sample. Tuberculous cavities may contain 107–109 activelymultiplying organisms. Sample preparation and assay protocols involve common laboratory practices and could easily and quickly be learned and implemented by laboratory staff. The RAPTOR is user friendly and could be used by medical examiner staff at time of need. The biosensor assay may also have potential applications for use on biopsy tissue from living individuals who have heavy pulmonary or disseminated infections by M. tuberculosis.
ABSTRACT A total of 300 strains of lactic acid bacteria (LAB) were screened for the production of bacteriocins active against Listeria innocua or Escherichia coli by using two different techniques: the conventional well‐diffusion assay and a newly developed one based on turbidity measurement of the growth of an indicator bacterium in the neutralized cell‐free supernatant of the putative bacteriocin‐producing strain. The latter technique, designated as the multiwell antagonistic activity assay (MW3A), offers advantages over the previously known methods. Notably, it allows testing simultaneously a large number of LAB for the production of bacteriocins against more than one indicator strain, while including appropriate controls for confirmation of the bacteriocinogenic nature of the inhibitory substances. Furthermore, five enterococcal strains were shown to produce bacteriocins active against the gram‐positive and gram‐negative indicator strains used in this study, suggesting that these enterococci or their bacteriocins have good potential to enhance food safety and keeping quality. PRACTICAL APPLICATIONS The present study describes a rapid and semiautomatic nephelometry method to screen for bacteriocin‐producing lactic acid bacteria (LAB). The method uses a microtiter plate where the inhibition of the growth of indicator strains in the presence of a bacteriocin is revealed by a decrease in OD as function of time. Concomitantly, this method determines the proteinaceous nature of the antimicrobial substance and its mode of action (i.e., bactericidal or bacteriostatic) while excluding the effect of interfering antimicrobials produced by LAB (e.g., hydrogen peroxide and organic acids). Furthermore, the newly described method differs from previously described ones by its ability to test for a relatively large number of putative bacteriocin‐producing strains using different controls and indicator strains, and allow early detection of bacteriocin‐producing strains (within 24 h). Another feature of this method is its ability to detect with confidence bacteriocins active against gram‐negative bacteria. These performances have potential to be used for rapid detection of LAB having strong capacity to inhibit pathogenic or spoilage bacteria by means of bacteriocins for possible applications in food preservation strategies.
ABSTRACT Tuberculosis (TB) is a major cause of morbidity and mortality worldwide. One hundred and nineteen acid‐fast bacilli‐positive smears for Mycobacterium Growth Indicator Tube cultures from 119 patients were examined by microscopy for the presence of cord formation. The results were compared with those of the traditional TB identification method, IS6110 polymerase chain reaction (PCR), and the Capilia TB assay which uses a monoclonal antibody to identify. With the traditional TB identification method, 57 of these 119 specimens were determined to be positive for Mycobacterium tuberculosis complex, and the organisms in the remaining 62 specimens were identified as non‐tuberculosis mycobacteria (NTM). Both IS6110 PCR and the Capilia TB assay yielded results identical to those of the traditional method with 57 true TB and 62 NTM. For the cord formation assay, all 62 NTM cultures were negative, but 54 of the 57 true TB cultures were positive. Therefore, the cord formation method had a sensitivity of 94.74% (54/57), specificity of 100% (62/62), negative predictive value of 95.38% (62/65) and positive predictive value of 100% (54/54) for identification of M. tuberculosis complex. The cord formation method is less expensive and 3–5 weeks quicker than the biochemical tests in the identification of M. tuberculosis.PRACTICAL APPLICATIONSDue to the slow growth of Mycobacterium tuberculosis bacilli, delays in the detection of TB infection may occur in clinical TB laboratories when only conventional methods for recovery of mycobacteria are used. This problem can be supported by other techniques, such as cord formation in Kinyoun‐stained smears of Mycobacterium Growth Indicator Tube cultures and molecular biology‐based systems, which can be used in combination to obtain accurate results in a much shorter period of time.
ABSTRACT Vibrio cholerae O1 is natural to the aquatic environment and can cause gastrointestinal infections when it is consumed from contaminated bivalves. Under unfavorable conditions, this bacterium enters into a viable but nonculturable state. Immunofluorescence and polymerase chain reaction (PCR) methods were a useful alternative for detecting this microorganism without a pre‐enrichment step. We investigated the detection limit of the direct fluorescent antibody (DFA)‐direct viable count (DVC) and PCR techniques for the identification of V. cholerae O1 in mussel ( Mytilus edulis ) samples. When 10 3 cfu/mL V. cholerae O1 were inoculated in samples, 10 2 –10 3 bacteria mL −1 were determined by immunofluorescence tests and 67% of the samples were positive by PCR assay. No significant difference ( T statistic value = 6.5, P = 0.2049) between DFA and DFA‐DVC procedures was observed. No presence of endogenous V. cholerae O1 was detected . PRACTICAL APPLICATIONS Vibrios are considered the major cause of identifiable illness and death from shellfish consumption. In Argentina, the viable but nonculturable (VBNC) forms of Vibrio cholerae O1 were identified in samples of water and plankton. Because of these facts, it is relevant to research the presence of V. cholerae O1 in aquatic bivalves. Immunofluorescence and polymerase chain reaction methods are a useful alternative to traditional enrichment testing for detecting both culturable and VBNC forms of V. cholerae O1. In this work, it was demonstrated that these methods were sensitive and efficient for detecting V. cholerae O1 in mussels without a pre‐enrichment step. Moreover, they can be a useful tool for the rapid detection of this pathogen in the seafood industry.
ABSTRACTPseudomonas genus‐specific primers targeting the 16s rRNA gene were used in a real‐time polymerase chain reaction (PCR) assay for rapid analysis of Pseudomonas isolated from retail chicken carcasses. A multiplex PCR assay was also designed using specific primers targeting the gyrase B sub‐unit gene to rapidly distinguish between several species of poultry significant Pseudomonads. The assays were used to evaluate the species and level of spoilage Pseudomonads on raw chicken carcasses over an 8‐day storage period. No Pseudomonas were detected on the chicken carcasses until 4 days after storage using culturing and plating techniques, but the PCR‐based assays developed in this research were more sensitive and detected Pseudomonas in carcass rinses performed immediately after processing. With the multiplex PCR assay, it was determined that most of the Pseudomonas spoilage was because of Pseudomonas fluorescens and Pseudomonas fragi.PRACTICAL APPLICATIONSEconomic losses as a result of spoilage are estimated between 5 and 17 billion dollars annually. In the poultry industry, the Pseudomonads not only cause the majority of this spoilage, but are also primary biofilm formers which may harbor and spread pathogens. Currently, only biochemical assays are available in the food industry for detection and differentiation of Pseudomonads. However, biochemical assays require 5 days for results, are limited to detection of a few species, and often produce erroneous results. The PCR assays developed in this work can reduce detection time to a few hours. Furthermore, these nucleic acid‐based assays have the potential to simplify the identification process, reduce food spoilage and foodborne illness, and speed the diagnosis of ill birds.
