Journal of Rapid Methods and Automation in Microbiology via MedWorm.com

Modification of fung double tube and cp anaselect oxyplate methods to improve their performance in enumerating clostridium perfringens from sewage and environmental waters

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

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. (Source: Journal of Rapid Methods and Automation in Microbiology)

An innovative microplate assay to facilitate the detection of antimicrobial activity in plant extracts

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

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 valu...

USE OF LOOP-MEDIATED ISOTHERMAL AMPLIFICATION ASSAY FOR THE DETECTION OF VIBRIO ANGUILLARUM O2β, THE CAUSATIVE AGENT OF VIBRIOSIS IN ATLANTIC COD, GADUS MORHUA

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

A loop-mediated isothermal amplification (LAMP)-based assay for the detection of Vibrio anguillarum O2[beta], 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 O2[beta] 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 sen...

Polymerase chain reaction-based assays for the detection and differentiation of poultry significant pseudomonads

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

Pseudomonas 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 ...

LOOP-MEDIATED ISOTHERMAL AMPLIFICATION – AN ASSAY FOR THE DETECTION OF ATYPICAL FURUNCULOSIS CAUSED BY AEROMONAS SALMONICIDA IN ATLANTIC COD, GADUS MORHUA

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

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[minus]1, whereas there was a decrease in detection limit by 1 log dilution in the presence of mucus as inhibito...

Simple and rapid method for detecting foodborne shigella by a loop-mediated isothermal amplification

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

A novel method was developed and evaluated for detecting Shigella by a loop-mediated isothermal amplification (LAMP) in this paper. Specific primers were specially designed and selected for recognizing seven distinct sequences of ipaH gene, which was performed only by Shigella. The sensitivity of the LAMP assay was 43 cfu/mL, approximately equal to that of real-time polymerase chain reaction assay. The LAMP assay developed in this study is specific, sensitive and suitable for the detection of foodborne Shigella. The LAMP method reported here provided a powerful tool for detection of foodborne Shigella due to its specificity, sensitivity and rapidity. (Source: Journal of Rapid Methods and Automation in Microbiology)

Development of a spot-titer culture assay for quantifying bacteria and viral indicators

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

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 (R2 = 0.989 for laboratory strains and R2 = 0.972 for environmental samples), while more cost- and labor-efficient. 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 mat...

Multiplex pcr for direct identification of thermophilic campylobacter, c. jejuni, c. coli, c. lari and c. upsaliensis and simultaneous detection of cdtb gene

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

A multiplex polymerase chain reaction (PCR) assay has been developed for the identification of the four thermophilic Campylobacter commonly associated with human gastroenteritis including C. jejuni, C. coli, C. lari and C. upsaliensis. The best combination of primers and the annealing temperature of multiplex PCR were examined. Detection limit was 2 × 105 cfu and 100 ng of DNA or whole-cell suspension. The multiplex PCR was applied for the direct detection and differentiation of Campylobacter species in 33 human and 45 chicken caeca isolates. Of the 78 specimens evaluated by the multiplex PCR, 55 (70.5%) were identified as C. jejuni, 18 (23.0%) as C. coli and 5 (6.41%) as a mixed infection with both species. Comparison of hippurate test and multiplex PCR demonstrated five (6.41%) isolates...

PRODUCTION OF SHIGA-LIKE TOXINS BY ESCHERICHIA COLI O157 : H7: EFFECTS OF OTHER BACTERIA AND ANALOGS OF QUORUM SENSING MOLECULES

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

Accompanied with the growth of Escherichia coli O157 : H7, there was a production of Shiga-like toxins by this pathogenic bacterium. Time-course studies indicated that the accumulation of toxins in the medium occurred mainly at the stationary phase of cell growth. The growth of E. coli O157 : H7 in culture media was not significantly affected by the presence of other bacteria, e.g., E. coli K-12, E. coli B6, Salmonella and Pseudomonas, even at high ratios. However, the production of Shiga-like toxins by E. coli O157 : H7 could be reduced by certain other bacteria, e.g., E. coli K-12, Pseudomonas aeruginosa but not Pseudomonas putida. These lowering effects by other background bacteria on the toxin production were also observed in experiments using regular and irradiated ground beef. The pr...

Evaluation of monostaph plus in comparison to two other latex agglutination tests for the identification of staphylococcus aureus

Journal of Rapid Methods and Automation in Microbiology — Wed, 02 Dec 2009 00:00:00 +0100

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. (Source: Journal of Rapid Methods and Automation in Microbiology)

Fung's corner

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Dec 2009 00:00:00 +0100

(Source: Journal of Rapid Methods and Automation in Microbiology)

Fluorescent measurements of dna, rna and proteins to perform comparative analyses of microbial communities from the environments

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

Simple and rapid methods for the quantification of DNA, RNA and proteins using specific fluorescent dyes are proposed for the comparison and monitoring of microbial communities from the environment. The purpose of this study was the use of straightforward in situ methods which voided the need for preservation of samples and the risk of potential degradation and quantitative changes during transportation. Aside from this, methods used to obtain information on environmental microbial communities are generally time-consuming and present certain difficulty above all when working on solid substrates such as soils and rocks. New generation fluorescent dyes that bind specifically to DNA, RNA and proteins allow simple and rapid estimates of these biomolecules in crude environmental samples. Monito...

Suitability of intergenic spacer or internal transcribed spacer microsatellite-primed pcr for the identification of rhizoctonia solani and some phytofungi

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

The intergenic spacer (IGS) region or internal transcribed spacer (ITS) region were used in pair-combinations with microsatellite-primed polymerase chain reaction (MP-PCR) primers to establish whether additional polymorphisms can be yielded. A total of 24 Rhizoctonia solani isolates representing 13 anstomosis groups and 9 different fungal species isolate were recovered from different areas and hosts. Forty different primer combinations were tested for their ability to provide discrete bands and individual isolates' readily interpretable and reproducible IGS/ITS-MP-PCR profiles. Both approaches produced highly reproducible and complex genomic fingerprints, with fragments ranging in size from 100 to 2,000 bp (IGS-MP-PCR) and 50 to 2,000 bp (ITS-MP-PCR). MP-PCR markers yielded more bands than...

Persistence of salmonella spp. on chicken skin after exposure to an italian marinade*

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

A series of experiments with chicken skin was undertaken to determine the effect of an Italian marinade on persistence of Salmonella spp. during refrigerated storage and marinating. Chicken skin was inoculated with 0.4 to 3.7 log of multiple antibiotic resistant strains of Salmonella Typhimurium (n = 3), Kentucky (n = 1) or Hadar (n = 1). Chicken skin was then exposed to the Italian marinade for 4 or 24 h at 6C to simulate normal marinating conditions of consumers. The persistence of Salmonella spp. on chicken skin was reduced (P < 0.05) by the Italian marinade with a greater reduction observed at 24 h than at 4 h of marinating. As expected, the persistence during marinating increased as a function of the initial number of Salmonella inoculated. In general, the effect of the Italian marina...

Comparative analysis of methods for the purification of dna from low-biomass samples based on total yield and conserved microbial diversity

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

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. (Source: Journal of Rapid Methods and Automation in Microbiology)

Evaluation of cord formation in kinyoun-stained smears of mgit cultures as a rapid identification method for mycobacterium tuberculosis complex

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

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 m...

Determination of indicator bacteria in pharmaceutical samples by multiplex pcr

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

Rapid and sensitive detection techniques for indicator pathogens are important in pharmaceutical industry. However, common detection methods rely on bacterial culture in combination with biochemical tests, a process that typically takes 5[ndash]6 days to complete. Thus, the aim of this study was to develop a multiplex polymerase chain reaction (mPCR) assay for simultaneous detection and identification of four indicator pathogenic bacteria in a single reaction. Specific primers for indicator bacteria, namely Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosaand Salmonella, were applied to allow simultaneous detection of them, and the sensitivity and specificity of each primer pairs were determined. In the mPCR with mixed DNA samples, specific bands for corresponding bacteria we...

Multiplex detection of escherichia coli and salmonella enteritidis by using quantum dot-labeled antibodies

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

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[ndash]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[ndash]antibody conjugates used in labeling were optimized to enhance the sensitivity of the assay. After labeling bacteria with QDs, the quenching observed between bead[ndash]bacteria complex and QDs was e...

A new fluorogenic assay for monitoring and determining planktonic and biofilm forms of pseudomonas aeruginosa viable count in vitro

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

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 (R2 = 0.9487) and biofilm cells of P. aeruginosa (R2 = 0.9296). 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...

Direct fluorescent antibody-direct viable count and polymerase chain reaction detection limit for the identification of vibrio cholerae o1 in mussels (mytilus edulis)

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

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 103 cfu/mL V. cholerae O1 were inoculated in samples, 102[ndash]103 bacteria mL[minus]1 were determined by immunofluorescence tests and 67% of the samples were positive by PCR assay. No significant differe...

Application of a colony pcr technique with fung's double tube method for rapid detection and confirmation of clostridium perfringens

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

A colony polymerase chain reaction (PCR) technique was applied with an established Fung's double tube (FDT) method for rapid detection and confirmation of Clostridium perfringens. Published sequences of PCR primers for C. perfringens alpha toxin gene were used and PCR conditions were optimized. From the detection of C. perfringens by FDT tube to the confirmation by a colony PCR assay took as short as 16[ndash]18 h. The method was applied to 147 isolates of anaerobic sulfite reducing bacteria isolated from foods, sewages and animal clinical specimens. The results were compared with standard methods for the confirmation of C. perfringens. Of those 147 suspected isolates, 97 and 99 were confirmed as C. perfringens by standard methods and the colony PCR technique, respectively. We found the de...

A novel method for assessment of 16s rrna gene copy number in bacterial genomes by pulsed-field gel electrophoresis and pcr amplification

Journal of Rapid Methods and Automation in Microbiology — Tue, 01 Sep 2009 23:00:00 +0100

16S rRNA gene (rrn) copy number in bacterial genomes is indicative of ecological strategies of bacteria and is critical for quantification of bacterial abundance in mixed populations using rrn-based approaches. For accurate assessment of rrn copies, a novel technical strategy by means of pulsed-field gel electrophoresis and polymerase chain reaction amplification analysis was introduced. Experimental and in silico analysis on a test bacterial culture Caulobacter crescentus proved it to be simple, effective, accurate and a good alternative to traditional time-consuming methods. This method can be used for routine determination of gene copy number in most bacteria whose full genome sequences are not available. Moreover, the pulsed-field gel electrophoresis bands containing a target gene frag...

Editorial

Journal of Rapid Methods and Automation in Microbiology — Mon, 31 Aug 2009 23:00:00 +0100

(Source: Journal of Rapid Methods and Automation in Microbiology)

Nested automated ribosomal intergenic spacer analysis: a rapid and accurate method for comparison of bacterial community composition

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

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. Nested automated ribosomal int...

Large-volume filtration for recovery and concentration of escherichia coli o157:h7 from ground beef*

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

This report describes a novel three-stage filtration system based on a leukocyte removal filter, a glass fiber prefilter, and a membrane capture filter. Data are presented on factors (e.g., particle size, bacteria binding, pH) affecting filtration performance and protocol design. Escherichia coli O157:H7 at less than 1 cfu/g were quantitatively recovered from 10 g of stomached ground beef in 15 min, and detected on selective media within 24 h. The methodology presented in this study allows the rapid concentration of Escherichia coli O157:H7 from the large volumes of stomached ground beef. The primary application is in rapid biosensor detection and quantitation of low levels of pathogens in foods without enrichment. The isolated bacteria can be recovered in a volume of [sim]20 µL and are v...

A high throughput screening method for 1,3-dihydroxyacetone-producing bacterium by cultivation in a 96-well microtiter plate

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

1,3-Dihydroxyacetone (DHA) is used extensively in the cosmetic industry, and is the main active ingredient in all sunless tanning skincare preparation. In order to more efficiently and rapidly screen suitable strains or mutants for production of DHA, a high throughput screening method for DHA-producing bacterium by cultivation in a 96-well microtiter plate was developed. With this screening method, more than 100 strains that were able to convert glycerol to DHA were isolated from soil samples, and a mutant of Gluconobacter oxydans ZJB-605 that displayed the highest DHA productivity was obtained. The practical application of this work is to promote the microbial process for isolating DHA-producing bacterium and screening DHA-overproducing mutant. With it, DHA manufactory can improve efficie...

Serological attraction of nontoxic egg component to staphylococcal anti-enterotoxin

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

Raw whole liquid and dried eggs which were purported to contain staphylococcal enterotoxin were analyzed by a number of enzyme-linked immunosorbent assay (ELISA)-based methods. The initial evaluation was to establish whether the purported positive ELISA reactions were a result of toxin-anti-enterotoxin serological activity. A secondary consideration was to determine whether the putative protein occurred in the yolk and/or white portions of the eggs. A manual polyvalent detection system, manual monovalent ELISA and an automated polyvalent enzyme-linked fluorescent immunoassay were used to investigate this component in eggs. The ELISA results were instantaneous and showed strong positive reactions (false positives) with the manual and automated polyvalent systems, suggesting nonspecific bind...

Rapid detection of listeria monocytogenes in ground turkey by immunomagnetic separation and polymerase chain reaction

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

In this study rapid, specific and sensitive IMS method was used to determine the prevalence of L. monocytogenes in fresh ground turkey and PCR technique was used for the verification of the L. monocytogenes isolates. (Source: Journal of Rapid Methods and Automation in Microbiology)

Simultaneous direct detection of salmonella spp., listeria monocytogenes and escherichia coli o157 in milk samples by magnetic extraction and multiplex pcr

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

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 102 cells of each pa...

Comparison of three different methods for the isolation of bacteriocin-like inhibitory substances from bifidobacterium infantis bcrc 14602

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

The objective of this research was to compare three different methods for the isolation of bacteriocin-like inhibitory substances (BLIS) from Bifidobacterium infantis BCRC 14602 (Bioresource Collection and Research Center) for their ease of use and reliability. Ammonium sulfate with 80% saturation of the neutralized cell-free supernatant resulted in 80% of total activity and a 4.56-fold increase in specific activity. After dialysis, the specific activity increased 76-fold, but total activity recovered decreased to 8%. In the second method, adsorption and desorption of the BLIS onto/from the producer cells, the adsorption of the BLIS to the producer cells was strongly affected by the pH of the culture broth whereby 100% adsorption to the killed cells occurred between pH 6.0 and 7.0. Desorpt...

Identification of proteins involved in infectivity and enterotoxin production in enterobacter sakazakii

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

Enterobacter sakazakii is a gram-negative rod bacterium that was formerly known as "yellow-pigmented Enterobacter cloacae" until 1980. It is an emerging opportunistic pathogen associated with bacterial meningitis in immunocompromised neonates who ingested contaminated powdered infant formula. In several E. sakazakii-related outbreaks and sporadic cases, powdered infant formula was epidemiologically or microbiologically established as the source of infection. The International Commission on Microbiological Specifications for Foods has ranked E. sakazakii as a "severe hazard for restricted populations." However, the organism was isolated at very low levels from commercial powdered infant formula and dry environmental samples collected from infant formula factories. In most cases, the contami...

Rapid detection of brucella abortus by a novel proximity ligation-based loop-mediated isothermal amplification method

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

In this study, we present the novel proximity ligation-based loop-mediated isothermal amplification (P-LAMP) method for Brucella detection; this is the first time to combine the monoclonal antibody for identify microbe and LAMP method for high performance amplification DNA. The genomic DNA extraction procedure is not needed and a water-bath boiler is the only equipment required to complete the detection process. The P-LAMP method is useful for food safety pathogen detection in developing countries. (Source: Journal of Rapid Methods and Automation in Microbiology)

Identifying aceticlastic and hydrogenotrophic methanogens in psychrophilic and mesophilic granular sludges treating synthetic sewage by means of fish and cslm

Journal of Rapid Methods and Automation in Microbiology — Fri, 05 Jun 2009 04:00:00 +0100

In this study, temperature influence on aceticlastic and hydrogenotrophic methanogen groups was evaluated using FISH coupled with CSLM. (Source: Journal of Rapid Methods and Automation in Microbiology)

Development of multiplex pcr assays for detection of antimicrobial resistance genes in escherichia coli and enterococci

Journal of Rapid Methods and Automation in Microbiology — Mon, 01 Jun 2009 04:00:00 +0100

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. (Source: Journal of Rapid Methods and Automation in Microbiology)

Identification of cellulolytic bacteria isolated from the termite coptotermes curvignathus (holmgren)

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

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 ...

Rapid detection of germinating bacillus cereus cells using fluorescent in situ hybridization

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

This study presents an experimental design in order to find the best combination of germination conditions leading to a rapid and detectable fluorescent in situ hybridization (FISH) signal from Bacillus cereus spores present in pure cultures and milk samples. B. cereus ATCC 14579 and HER 1414 were incubated in 20 different growth media by using a combination of various germinants such as sugars, amino acids and dipicolinic acid. Also, three different germination factors were tested: incubation temperature, inoculum concentration and a heat shock treatment. Permeabilization procedure and hybridization time were optimized on the best germination condition found. B. cereus-specific FISH probes were validated under the optimized condition and in detection of spiked B. cereus spores in 1% ultra...

Identification of new target sequences for pcr detection of vibrio parahaemolyticus by genome comparison

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

A genome comparison method was used to identify specific target sequences for the polymerase chain reaction (PCR) detection of Vibrio parahaemolyticus, and the CDS value of this bacterium was compared with that of 139 other bacterial genomes. It was found that 20 CDS of V. parahaemolyticus were relatively specific according to their E value in BLAST (a new tool for comparing protein and nucleotide sequences), and four of them were selected for the design of PCR primers. There were positive amplification products of these four pairs of primers from nine V. parahaemolyticus strains, whereas there were no amplification products from nine other Vibrionaceae strains and four non-Vibrionaceae strains. An evaluation of detection sensitivities revealed that these four pairs of primers can be used ...

Development and evaluation of a loop-mediated isothermal amplification method for detecting escherichia coli o157 in raw milk

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

In the present study, we report the performance of a loop-mediated isothermal amplification (LAMP) assay detecting foodborne pathogen Escherichia coli O157. Three pairs of primers were specially designed for recognizing eight distinct sequences of rfbE gene. Time and temperature conditions for amplification of E. coli O157 were optimized to be 40 min at 65C. The LAMP assay gave artificially contaminated raw milk sample detection limit level of 410 cfu/mL which corresponds to three to five cells per reaction tube, while the detection level of conventional polymerase chain reaction was 4.1 × 104 cfu/mL. Data on naturally contaminated raw milk samples indicated that the LAMP method was highly specific and sensitive, giving 100% concordance with the ISO 16654: 2001 reference method. The loop-...

Enumerating chromogenic agar plates using the color qcount automated colony counter

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

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[ndash]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[minus]0.042. The mean log value differences between manual and automated counts demonstrated ...

Development of a multiwell antagonistic activity assay for the detection of bacteriocin production by lactic acid bacteria

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

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 substan...

Rapid detection of mycobacterium tuberculosis in lung tissue using a fiber optic biosensor

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

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 ob...

CHEF PROCEDURES: A RAPID HIGH-TEMPERATURE METHOD FOR SAMPLE PREPARATION, A HIGH VOLTAGE HEPES BUFFER SYSTEM AND THE USE OF NUSIEVE® AGAROSE

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

A rapid, high-temperature method of sample preparation from gram-negative bacteria for contour-clamped homogenous electric field (CHEF) electrophoresis is described, which utilizes a diethylpyrocarbonate nuclease inactivation step for preventing latent degradation of DNA. Also described is a simple 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer system that permits high-voltage CHEF electrophoresis in standard apparatus while preventing the strand scission events associated with tris(hydroxymethyl)aminomethane buffers. Finally, the use of NuSieve agarose in combination with standard analytical agarose was compared to specialty CHEF agarose, demonstrating the potential use of this reagent in CHEF gel separations. Contour-clamped homogenous electric field (CHEF) gel electrophoresis...

Simplified capacitance monitoring for the determination of campylobacter spp. growth rates

Journal of Rapid Methods and Automation in Microbiology — Mon, 02 Mar 2009 05:00:00 +0100

Capacitance monitoring is commonly used as an efficient means to generate growth curves of bacterial pathogens. However, the use of capacitance monitoring with Campylobacter spp. was previously determined to be difficult due to the complexity of the required media. We investigated capacitance monitoring using a simplified medium for the efficient and reproducible construction of growth curves for Campylobacter spp. It was determined that Campylobacter spp. should initially be propagated on Mueller Hinton plates in a microaerobic atmosphere (5% O2, 10% CO2, 85% N2) at 37C for 24 h, followed by transfer to Mueller Hinton biphasic cultures for 6 h at 37C in a microaerobic atmosphere. Serial dilutions of the Campylobacter spp. cultures should be used for inoculation of Bactometer wells that co...

Editorial

Journal of Rapid Methods and Automation in Microbiology — Sun, 01 Mar 2009 05:00:00 +0100

(Source: Journal of Rapid Methods and Automation in Microbiology)

Molecular characterization of listeria monocytogenes strains harboring listeria innocua putative transcriptional regulator gene lin0464