Colicin V38 and microcin C38 produced by Escherichia coli strain 38.

Escherichia coli strain 38, an isolate from turkeys, has been previously shown to produce one or more broad-spectrum bacteriocins against other related enteric bacteria. Using a collection of E. coli strains that synthesized well-characterized colicins or microcins, along with a set of colicin/microcin-insensitive mutants, we were able to classify the bacteriocins produced by strain 38. We determined that strain 38 produced a microcin (microcin C38) and a colicin (colicin V38) and that the amount of microcin C38 depended on the type of media on which it was grown. Escherichia coli strain 38 was found to have cross-immunity with the microcin C7-producing strain MC4100 and with the colicin V-producing strain 4674. OmpF mutant cells were found to be insensitive to microcin C38, whereas colicin V38 was not active on cells that had a cir mutation. Both microcin C38 and colicin V38 were inactivated by proteases. Microcin C38 was stable at extremes of pH (pH 1.5 and pH 13) and heat (10 min at 98 C) conditions, whereas colicin V38 was not. In addition, microcin C38 was found to be active against a broader spectrum of gram-negative bacteria than was colicin V38.

Culture variability associated with the U.S. Environmental Protection Agency Tuberculocidal Activity Test Method.

Tuberculosis continues to be a major world health threat. The etiologic agent is among the vegetative organisms most resistant to chemical disinfection. Tuberculocidal efficacy testing for regulatory approval of chemical germicides has evolved considerably over the past decade. A method currently in use is the Environmental Protection Agency Tuberculocidal Activity Test Method, a suspension test using a Mycobacterium bovis culture grown under specific conditions and stored frozen until used. Differing tuberculocidal label claims on products with similar formulations have raised questions concerning the equivalence of test suspensions prepared by different laboratories. Five M. bovis suspensions from laboratories currently performing this test were compared against a battery of three disinfectants at a single test site. A significant difference between test cultures was found, with two of the five exhibiting a significant difference from the other three and also from each other. There was a significant culture-by-disinfectant interaction, indicating that the five cultures did not respond in a consistent manner across the different disinfectants used. However, these differences were due to cultures that were not prepared in accordance with the standard procedure or otherwise did not meet the test suspension criteria. In addition, a 0.55% sodium hypochlorite solution was found to be a sensitive indicator of culture variability. These data reinforce the need to adhere to published procedures and guidelines when growing and preparing a tuberculocidal test suspension and shed light on the variables associated with this type of testing.

Staphylococcosis of turkeys. 6. Development of penicillin resistance in an interfering strain of Staphylococcus epidermidis.

Staphylococcus epidermidis strain 115, used as an interfering agent to help reduce the incidence of staphylococcosis in turkeys, was converted into a penicillin- and chloramphenicol-resistant strain designated 115R. This was accomplished by introducing a plasmid carrying the beta-lactamase (penicillinase) and chloramphenicol-resistance genes into S. epidermidis 115 by electroporation. The resultant strain, 115R, was an efficient producer of beta-lactamase and had marked increased resistance to penicillin and chloramphenicol. A beta-lactamase DNA probe was used to confirm the presence of the beta-lactamase gene in strain 115R. S. epidermidis strain 115R retained the characteristics of tissue adherence, bacteriocin production, and non-virulence that were present in the original non-transformed strain 115, and in addition should theoretically remain colonized in poults following treatment with penicillin.

Antimicrobial activity of environmental surface disinfectants in the absence and presence of bioburden.

Thirty-nine products representing six categories of disinfectants (alcohols, chlorines, dilute glutaraldehydes, iodophors, phenolics, and quaternary ammonium compounds) were first tested in the absence of bioburden, using four test methods with five test organisms. Products that performed best were retested with the same methods and organisms in the presence of both serum and whole blood, using 3- and 10-minute contact times. Only products containing high ethyl alcohol had consistently high antimicrobial activity regardless of the test method, test organism, or contact time used both in the absence and presence of bioburden. Although these specific formulations demonstrated ability to penetrate and inactivate high concentrations of microorganisms within heavy bioburden, optimum disinfection of environmental surfaces is highly formulation dependent. Other products tested showed deficiencies that contraindicate their use as environmental surface disinfectants in clinical dental settings.

RNA-DNA hybridization analysis of transcription of the plasmid ColV-K30 aerobactin gene cluster.

Plasmid pABN1 contains the genetic determinants for the aerobactin iron uptake system of plasmid ColV-K30. Transposon Tn1000 mutants of pABN1 defective in synthesis of a 50,000-dalton polypeptide were found neither to secrete nor to accumulate aerobactin, but were not impaired in iron transport functions, clearly indicating a role for this polypeptide in aerobactin biosynthesis. RNA-DNA hybridization studies with probes spanning the entire aerobactin gene cluster showed that the system is regulated at the transcriptional level by the availability of iron in the external medium. When induced by low-iron stress, all five genes of the cluster were transcribed at a uniformly high level. When repressed by excess iron, transcripts of the four biosynthesis genes were some 30-fold less abundant in the case of the parental ColV-K30 plasmid and 10-fold less for the recombinant plasmid pABN1, whereas the receptor gene in either plasmid was transcribed at only about a third of the induced level.

Comparison of the Scott Selecticult-HSV kit with conventional culture and direct immunoperoxidase staining for detection of herpes simplex virus in cultures of clinical specimens.

In a comparative study, clinical specimens were cultured for herpes simplex virus (HSV). The presence of virus was noted by the appearance of characteristic cytopathic effect, as determined by standard direct immunofluorescence techniques, by using a direct immunoperoxidase stain for viral antigen, or by using the Selecticult-HSV (SC-HSV) stain for viral antigen. There was 100% correlation between the SC-HSV stain and immunofluorescence staining in recognizing HSV-infected cells (81 of 81 positive specimens). In comparison with observation of cytopathic effect, the SC-HSV system and conventional culture detected 93 and 78% of positive cultures at 48 h postinoculation and 76 and 32%, respectively, at 24 h. By 5 days postinoculation, SC-HSV detected 100% of the positive specimens. As compared with the direct immunoperoxidase stain, SC-HSV stain was slightly more sensitive and gave less background stain. HSV serotypes 1 and 2 were both detected by the SC-HSV stain. The Scott SC-HSV kit appears to be an effective system for the diagnosis of HSV infections.

Evaluation of the virocult transport tube for isolation of herpes simplex virus from clinical specimens.

Herpes simplex virus survived in Virocult transport tubes and had a half-life of 3.5 days at 2 degrees C and 2.75 days at 22 degrees C. Of 2,000 consecutive clinical specimens transported on Virocult tubes and cultured for herpes simplex virus, 448 (22.4%) were positive. Comparison of the holding times between positive and negative cultures, up to 12 days, revealed no significant loss of positive cultures with time.

Characterization of snap-back RNAs in vesicular stomatitis defective interfering virus particles.

VSV defective interfering particles of various sizes and from several independent sources frequently contain plus and minus strand RNA. In many cases some of the complementary strands are covalently linked as snap-back molecules. Infectious particles on the other hand package little or no plus strands. Snap-back molecules from the three different sources examined so far vary in size but appear to conform to the same overall linear duplex structure with cross-links at the ends only. They each contain a base sequence which is a subset of the next larger one and appear to correspond to unique sequences in the L cistron of the genome. Possible origins for these snap-back molecules are discussed.

Inverted complementary terminal sequences in single-stranded RNAs and snap-back RNAs from vesicular stomatitis defective interfering particles.

Complementary single-stranded RNAs from three independent VSV defective interfering particle (DI) sources examined can anneal and give rise to monomeric and multimeric circular and linear double-stranded structures observable by electron microscopy under aqueous conditions. When the RNA from the shortest of these DI is spread from 80% formamide solutions, as many as 32% of the molecules are circular, suggesting that the single-stranded RNAs contain inverted complementary terminal sequences. This is strongly supported by the isolation of the putative terminal sequences which rapidly become RNase resistant base-paired structures after melting and quick-cooling the RNA. RNase digestion yields a major and a minor component, 60 to 70 and 135 to 170 nucleotides long respectively. Snap-back DI RNAs also contain inverted complementary sequences at both ends of the plus and minus strands of the duplexes since nicking these at the ends gives rise to double-stranded molecules which can form monomeric and multimeric circular and linear molecules. Thus, snap-back molecules most likely contain a covalent linkage between or near complementary terminal sequences on the two complementary strands as schematically shown in Fig. 5D.

Purification and characterization of covalently closed replicative intermediates of ColEl DNA from Escherichia coli.

Pulse-labeled ColEl DNA molecules, undergoing replication in Escherichia coli cells either in the absence or presence of chloramphenicol, were extracted and purified by neutral sucrose density gradient sedimentation and equilibrium centrifugation in an ethidium bromide-cesium chloride gradient. In the dye-buoyant density gradient, the replicating molecules were found in regions between the supercoiled and open-circular nonreplicating plasmid DNA, as well as in the open-circular region. In a neutral sucrose gradient, peaks of pulse label were found in the region of 26 to 38 S as well as at the 23 and 17 S positions corresponding to the positions of supercoiled and open-circular ColEl DNA. In alkaline sucrose gradient, nascent ColEl DNA was found to sediment as discrete peaks corresponding to 5-6, 7-9, and 14-16 S, indicating that at least one growing strand of the replicating molecule is produced discontinuously. In the electron microscope, many of the molecules appeared as partially supercoiled structures containing two open-circular branches of equal length, of less than 20% to more than 90% replicated. Branched open-circular molecules were not observed to any significant extent without prior treatment to induce single-strand scissions. The parental strands of the replicating molecules were determined to be covalently closed, but the superhelical density of the DNA was shown to be progressively decreased as replication proceeded.

Isolation of circular deoxyribonucleic acid from Salmonella typhosa hybrids obtained from matings with Escherichia coli Hfr donors.

Heterozygous, partial diploid Salmonella typhosa hybrids obtained from matings with Escherichia coli K-12 Hfr strains were observed to contain supercoiled, circular deoxyribonucleic acid (DNA) when examined by the dye-buoyant density method. Examination of one such S. typhosa hybrid after its loss, by segregation, of the inherited E. coli genetic markers revealed a concurrent loss of its supercoiled circular DNA. Subsequent remating of this segregant with various E. coli Hfr strains resulted in the reappearance of the circular DNA. Molecular weight determinations of circular DNA molecules isolated from a number of S. typhosa partial diploid hybrids were made by sucrose density gradient ultracentrifugation and electron microscopy. These studies revealed a range of molecular sizes among the various hybrids examined, but each hybrid exhibited only a single characteristic size for its contained circular DNA. The range of size is consistent with the presence in each hybrid of a different length of E. coli chromosome. It was concluded that the E. coli Hfr genetic segments transferred to these S. typhosa hybrids were conserved, in the diploid state, in the form of supercoiled, circular DNA molecules.