Rapid procedure for the detection of plasmids in Staphylococcus epidermidis.

A rapid, reproducible, mini-volume assay capable of detecting staphylococcal plasmid DNA in the range of 0.8 to 32 megadaltons has been developed. The assay employs lysostaphin-mediated lysis of cells followed by a short, low-speed centrifugation and does not require treatment with ribonuclease or protease or deproteinization with phenol. A period of only 24 h may be required to detect the presence and size of a plasmid once an organism has been isolated. This method has been used to study the plasmid ecology of Staphylococcus epidermidis and to correlate the presence or absence of plasmids with tetracycline, chloramphenicol, neomycin, penicillin, and cadmium resistances.     

Rapid procedure for isolation and detection of Salmonella plasmids

A rapid procedure is described for the isolation of high quality and quantity plasmid DNA from Salmonella. Plasmids of molecular mass ranging from 10 kbp to 147 kbp were isolated. The method yielded enough DNA from the low copy number virulence plasmids of various Salmonella serovars for visualization on an agarose gel. The method is based on the classic alkaline lysis methods, employing the same reagents, but without any incubation steps. Isolation of plasmid DNA was also attempted with the use of rapid DNA isolation kits, but without success. It was reasoned that endonucleases, present in the resin, might have degraded the plasmids. © Rapid Science Ltd. 1998     

Rapid procedure for detection and isolation of large and small plasmids.

Procedures are described for the detection and isolation of plasmids of various sizes (2.6 to 350 megadaltons) that are harbored in species of Agrobacterium, Rhizobium, Escherichia, Salmonella, Erwinia, Pseudomonas, and Xanthomonas. The method utilized the molecular characteristics of covalently closed circular deoxyribonucleic acid (DNA) that is released from cells under conditions that denature chromosomal DNA by using alkaline sodium dodecyl sulfate (pH 12.6) at elevated temperatures. Proteins and cell debris were removed by extraction with phenol-chloroform. Under these conditions chromosomal DNA concentrations were reduced or eliminated. The clarified extract was used directly for electrophoretic analysis. These procedures also permitted the selective isolation of plasmid DNA that can be used directly in nick translation, restriction endonuclease analysis, transformation, and DNA cloning experiments.     

A rapid screening procedure for staphylococcal plasmids

A method has been developed for rapidly screening representatives of all currently recognized species of the genus Staphylococcus for the presence of plasmid DNA. The isolated plasmid DNA is substantially free from contaminating chromosomal and relaxed plasmid DNA. The method will detect plasmids in strains grown on various types of solid or liquid culture media and is convenient enough for routine epidemiological studies.     

A simple procedure for isolation of cloning vectors and endogenous plasmids from viridans group streptococci and Staphylococcus aureus.

Isolation of plasmid DNA from viridans group streptococci is difficult, and preparations are often heavily contaminated with chromosomal DNA. We developed a simple protocol to isolate pure plasmid DNA for use in different molecular techniques, including automated sequencing. The protocol is also applicable for plasmid isolation from Staphylococcus aureus. In addition, the protocol allows isolation of pure endogenous plasmids from streptococci and S. aureus.     

Rapid method for the detection of group B streptococci from human sources

A modification of the classical CAMP test has been devised for the rapid detection of streptococci of serological Group B from human sources. The method was compared with detection based on the development of orange pigmented colonies on a starch-based medium and with detection by conventional methods. In a survey of vaginal carriage of Group B streptococci in parturient women, the modified CAMP test detected a carriage rate of 13.09%, the starch-based, pigment enhancing medium, 5.76% and the conventional methods, 8.38%. It proved to be particularly useful for detecting the organisms in the presence of other bacteria.     

Rapid method for the detection of Group B streptococci from human sources

A modification of the classical CAMP test has been devised for the rapid detection of streptococci of serological Group B from human sources. The method was compared with detection based on the development of orange pigmented colonies on a starch-based medium and with detection by conventional methods. In a survey of vaginal carriage of Group B streptococci in parturient women, the modified CAMP test detected a carriage rate of 13.09%, the starch-based, pigment enhancing medium, 5.76% and the conventional methods, 8.38%. It proved to be particularly useful for detecting the organisms in the presence of other bacteria.     

Functional properties of plasmids in lactic streptococci

Plasmid biology has become an important area of investigation in dairy starter cultures since it now appears that some properties, vital for successful milk fermentations, are coded by genes located on plasmid DNA. Some of these metabolic properties observed in lactic streptococci have been clearly established as being plasmid-mediated. Examples would be lactose utilization and in Streptococcus lactis subsp. diacetylactis the ability to produce a bacteriocin-like substance. Phenotypic and physical evidence for plasmid linkage has been obtained for other traits such as citrate, sucrose, galactose, glucose, mannose, and xylose utilization, proteinase activity, modification/restriction systems, as well as for nisin production. Further genetic evidence is now needed to confirm plasmid association to these properties. For some characteristics the association with plasmids is highly speculative and is solely based on the phenotypic loss of a metabolic property. In this category would be sensitivity to agglutinins, sensitivity to the lactoperoxidase-thiocyanate-hydrogen peroxide inhibitory system, arginine hydrolysis, and slime production. Other properties which appear plasmid-mediated in lactic streptococci and which will be discussed include inorganic ion resistance, drug resistance, and diplococcin production.     

Rapid screening method for the detection of antimicrobial substances

Bioluminescence is phenomenon where living organisms produce light and this production is directly dependent on metabolic activity of the organism. Genes encoding enzymes, luciferases, responsible for light production can be cloned into indicator strains, thus allowing sensitive detection of antimicrobial activity. This study utilized bacterial luciferase genes cloned into Staphylococcus aureus, Escherichia coli and Salmonella enterica serovar Typhimurium indicator strains and showed that the detection of antimicrobial activity can be obtained already in 2 h without laborious plate counting and overnight incubation. Indicator strains used in the study harboured luxAB genes responsible of producing light as well as luxCDE genes for synthesis of long-chain fatty aldehyde as substrate for light production. As a consequence, no exogenous aldehyde addition was needed allowing stable light production. Furthermore, the method was used for the detection of antimicrobial activity from lactic acid bacteria after the effect of organic acids was eliminated.     

Conjugative R plasmids in group C and G streptococci.

Two streptococcal isolates of groups C and G harbored conjugative R plasmids with molecular weights of 17 X 10(6) (pIP646) and 20 X 10(6) (pIP920). These plasmids carried genetic markers for resistance to macrolides and related drugs, as well as to chloramphenicol (pIP920), and have very similar HindIII restriction enzyme patterns.     

Marker rescue allows direct selection for recombinant plasmids in streptococci

Summary Resident deletion derivatives (Em^s or Cm^s) of the streptococcal plasmid vector pGB301 rescue antibiotic resistance genes from linearized pGB301 (Em^r, Cm^r) DNA with high frequency. Insertion of passenger DNA next to an antibiotic resistance determinant of pGB301, which is missing on the resident plasmid, forces corescue of these two plasmid domains, thus allowing direct selection for recombinant plasmids.     

Rapid screening procedure based on headspace solid-phase microextraction and gas chromatography-mass spectrometry for the detection of many recreational drugs in hair

An increasing number of synthetic drugs are appearing on the illicit market and on the scene of drug use by youngsters. Official figures are underestimated. In addition, immunochemical tests are blind to many of these drugs and appropriate analytical procedures for routine clinical and epidemiological purposes are lacking. Therefore, the perceived increasing abuse of recreational drugs has not been proved yet. In a previous paper, we proposed a procedure for the preliminary screening of several recreational substances in hair and other biological matrices. Unfortunately, this procedure cannot apply to cocaine. Consequently, we performed a new headspace solid-phase microextraction and gas chromatography-mass spectrometry (HS-SPME-GC-MS) procedure for the simultaneous detection of cocaine, amphetamine (A), methamphetamine (MA), methylen-dioxyamphetamine (MDA), methylen-dioxymethamphetamine (MDMA), methylen-dioxyethamphetamine (MDE), N-methyl-1-(1,3-benzodioxol-5-yl)-2-butanamine (MBDB), ketamine, and methadone in human hair. Hair was washed with water and acetone in an ultrasonic bath. A short acid extraction with 1M hydrochloric acid was needed; the fiber was exposed to a 5 min absorption at 90 degrees C and thermal desorption was performed at 250 degrees C for 3 min. The procedure was simple, rapid, required small quantities of sample and no derivatization. Good linearity was obtained over the 0.1-20.0 ng/mg range for the target compounds. Sensitivity was good enough: limits of detection (LOD) were 0.7 ng/mg of hair for the majority of substances. The intra-day precision ranged between 7 and 20%. This paper deals with the analytical performance of this procedure and its preliminary application to hair samples obtained on a voluntary basis from 183 young people (138 males and 45 females) in the Rome area.     

Vector plasmids and the strategy of molecular cloning for streptococci

The experiments on elaboration of gene engineering methodology for streptococci are described. Two vectors were constructed for DNA cloning in streptococci on the basis of plasmids pSM19035 and pIP2501. Some of the plasmids occurred to be valuable vectors for molecular cloning in bacilli. Peculiarities of the transformation mechanism in streptococci were found to impede the molecular cloning. The recombination technique of cloning was successfully used in the streptococcal system.     

Rapid extraction of plasmids from Clostridium perfringens.

Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens. The first method involved lysis of 1 to 2 ml of C. perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl. DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel. The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS). Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel. Of 57 strains of C. perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method. These new plasmids were of higher molecular mass and ranged up to 68 megadaltons. Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA. An additional 159 isolates of C. perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.     

Rapid detection of salmonella in foods—a convenient two-day procedure

A new method for detecting salmonellas in foods within 42 h is described. This highly specific and sensitive selective motility procedure gave an efficiency of 96.8% when tested against traditional methods using more than 800 food samples. It is stable at ambient temperatures and can be prepared for use in 5 min.     

Preenrichment procedure for detection of Salmonella in gelatin.

Trypticase soy broth was superior to nutrient and lactose broths as a preenrichment medium for the detection of Salmonella in artificially and naturally contaiminated gelatin. The detection rate for Salmonella were further enhanced when homogenization of the gelatin-broth mixture was accomplished by the use of gelatinase rather than by heating of 45 degrees C. Detection rats were also increased by adjusting the pH of the gelatin-broth mixture of 7.0, optimum pH for gelatinase (EC 3.4.23.2) activity.