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At least 217 records · Page 12Linked to original sources
PubMed2005

Presence of multidrug-resistant enteric bacteria in dairy farm topsoil.

In addition to human and veterinary medicine, antibiotics are extensively used in agricultural settings, such as for treatment of infections, growth enhancement, and prophylaxis in food animals, leading to selection of drug and multidrug-resistant bacteria. To help circumvent the problem of bacterial antibiotic resistance, it is first necessary to understand the scope of the problem. However, it is not fully understood how widespread antibiotic-resistant bacteria are in agricultural settings. The lack of such surveillance data is especially evident in dairy farm environments, such as soil. It is also unknown to what extent various physiological modulators, such as salicylate, a component of aspirin and known model modulator of multiple antibiotic resistance (mar) genes, influence bacterial multi-drug resistance. We isolated and identified enteric soil bacteria from local dairy farms within Roosevelt County, NM, determined the resistance profiles to antibiotics associated with mar, such as chloramphenicol, nalidixic acid, penicillin G, and tetracycline. We then purified and characterized plasmid DNA and detected mar phenotypic activity. The minimal inhibitory concentrations (MIC) of antibiotics for the isolates ranged from 6 to >50 microg/mL for chloramphenicol, 2 to 8 microg/mL for nalidixic acid, 25 to >300 microg/mL for penicillin G, and 1 to >80 microg/mL for tetracycline. On the other hand, many of the isolates had significantly enhanced MIC for the same antibiotics in the presence of 5 mM salicylate. Plasmid DNA extracted from 12 randomly chosen isolates ranged in size from 6 to 12.5 kb and, in several cases, conferred resistance to chloramphenicol and penicillin G. It is concluded that enteric bacteria from dairy farm topsoil are multidrug resistant and harbor antibiotic-resistance plasmids. A role for dairy topsoil in zoonoses is suggested, implicating this environment as a reservoir for development of bacterial resistance against clinically relevant antibiotics.

Anti-Bacterial Agents↗
PubMedDate not supplied

Emergence of sulfamethoxazole-trimethoprim resistant Shigella flexneri in Northeastern Brazil.

In contrast to prior experience in this setting, three of four Shigella flexneri strains recently isolated from patients in Northeastern Brazil with acute inflammatory diarrhea were found to be resistant to sulfamethoxazole, trimethoprim and the combination in vitro. We performed mating studies to determine if the resistance was transferable, and then isolated and characterized plasmid DNA from the resistant Shigella isolates, other resistant Enterobacteriaceae isolated simultaneously from the stools of these individuals, and transconjugant strains. Each of the resistant Shigella strains contained a large plasmid. These plasmids were of different molecular weights ranging from 30 to 50 Mdal in size. Two of these plasmids were transferred with sulfamethoxazole-trimethoprim resistance to E. coli K-12 recipient strains. These findings of transferable resistance to sulfamethoxazole-trimethoprim associated with plasmids in Shigella and in other Enterobacteriaceae raises concerns about the potential limitations of this widely used antimicrobial combination.

DNA, Bacterial↗
PubMedDate not supplied

[Concern of the bacteriology laboratory in the diagnosis and surveillance of gonococcal and meningococcal infections].

The usual laboratory of bacteriology has an essential role in isolation, bacteriological diagnostic and testing of resistance towards antibiotics of gonococcal infections. Searching of beta-Lactamase will be carried out when inhibition diameters around penicillinase C disks will be inferior to 18 mm. The specialized laboratory will bring along useful information through the study of epidemiologic markers: auxotyping, plasmid characterization (connection with african types (2.6 and 3.7 M-daltons) and with South-East Asia (2.6; 4.5 and 24.5 M-daltons) and eventually serotypings by using proteins from outer-membrane. A similar action will be taken by all laboratories of bacteriology: isolation, identification, susceptibility searching and serogrouping from meningococci isolated from rhinopharynx and from C.S.F. or from blood culture. Eight groups whose specificities are carried by capsular polysaccharides are actually described (A, B, C, X, Y, Z, 29E and W135). The electrosyneresis will be useful when bacteria will not be possible to isolate. Serotyping techniques whose importance is essential are still depending on specialized laboratory.

Clinical Laboratory Techniques↗
PubMed1986

A new mechanism of plasmid trimethoprim resistance. Characterization of an inducible dihydrofolate reductase.

The dihydrofolate reductase encoded by plasmid pUK1123, which confers only a moderate level of trimethoprim resistance on its host, has been isolated and characterized. This enzyme, designated type IV, differs markedly from all previously described plasmid dihydrofolate reductases. It has a relatively high molecular weight of 46,700 as measured by gel filtration and, unlike previous plasmid dihydrofolate reductases, its synthesis is induced in the presence of increasing concentrations of trimethoprim. It is only slightly resistant to trimethoprim but is competitively inhibited by this drug with an inhibitor binding constant of 63 nM. In addition, the enzyme has a relatively low affinity for the substrate, dihydrofolate (Km = 37 microM). This is the first report of a plasmid trimethoprim resistance mechanism resulting from the induced synthesis of a large molecular weight dihydrofolate reductase which is only slightly resistant to trimethoprim. The possible origins of the type IV enzyme are discussed.

Chromatography, Gel↗
PubMed1986

Genetic and physical characterization of IncM plasmid pBWH1 and its variance among natural isolates.

We present a genetic and physical characterization of the IncM plasmid pBWH1. A physical map was constructed for the enzymes EcoRI, BamHI, SalI, BglII, HindIII, MstII, and XhoI. A series of deletions and a series of subclones of pBWH1 were constructed and used to determine the locations on this map of the transfer region; the replication region; and the genes determining resistance to beta-lactams, chloramphenicol, the sulfonamides, and gentamicin. We compared 51 different isolates, including isolates which had lost individual antibiotic resistances or the transfer phenotype, and showed that variations occurred in all regions of the plasmid genome. Frequently, correlations could be made between phenotypic variation and variation of the EcoRI fragments which contained the gene determining that phenotype.

Chromosome Mapping↗
PubMed1990

Characterization of Propionibacterium plasmids.

Plasmid DNAs from 15 Propionibacterium strains were characterized by using restriction endonuclease analyses, DNA-DNA hybridizations, and curing experiments. Restriction endonuclease analysis identified seven distinct plasmids (pRGO1 through pRGO7). Detailed restriction maps were constructed for four of these plasmids. DNA-DNA hybridization analysis revealed that plasmids pRGO1 and pRGO2 had extensive sequence homology and that both were homologous to pRGO7 and to similar sequences of pRGO5. Plasmids pRGO4 and pRGO6 did not have any significant sequence homology with any of the other plasmids. Plasmid pRGO3 had partial sequence homology only with pRGO7. Curing of plasmids pRGO1, pRGO2, and pRGO5 was achieved by treatment with acriflavin, but we failed to identify any plasmid-encoded bacteriocin production, carbohydrate fermentation, or antibiotic resistance. However, physical evidence was obtained that tentatively linked the clumping phenotype of Propionibacterium jensenii P38 with plasmid pRGO5.

Base Sequence↗
PubMed1980

Genetic and molecular characterization of R plasmids incompatible with R387 (IncK).

More than 70 conjugative R plasmids have been isolated from wild-type strains originating mainly in South-East Europe and identified as incompatible with the reference plasmid R387 of the incompatibility group K. These plasmids, governing different resistance patterns, have been characterized as P1 cotransducible species of between 50 and 60 mega-daltons. In contrast to the genetic similarity between all these plasmids and the reference type R387, their DNA revealed different digestion patterns after EcoRI treatment, although a number of common fragments could be identified. The molecular and genetic properties of these plasmid species demonstrate a phylogenetic relatedness.

DNA, Bacterial↗
PubMed1999

Molecular characterization of transforming plasmid rearrangements in transgenic rice reveals a recombination hotspot in the CaMV 35S promoter and confirms the predominance of microhomology mediated recombination.

The characterization of plasmid-genomic DNA junctions following plant transformation has established links between DNA double-strand break repair (DSBR), illegitimate recombination and plasmid DNA integration. The limited information on plasmid-plasmid junctions in plants comes from the dicot species tobacco and Arabidopsis. We analyzed 12 representative transgenic rice lines, carrying a range of transforming plasmid rearrangements, which predominantly reflected microhomology mediated illegitimate recombination involving short complementary patches at the recombining ends. Direct end-ligation, in the absence of homology between the recombining molecules, occurred only rarely. Filler DNA was found at some of the junctions. Short, purine-rich tracts were present, either at the junction site or in the immediate flanking regions. Putative DNA topoisomerase I binding sites were clustered around the junctions. Although different regions of the transforming plasmid were involved in plasmid-plasmid recombination, we showed that a 19 bp palindromic sequence, including the TATA box of the CaMV 35S promoter, acted as a recombination hotspot. The purine-rich half of the palindromic sequence was specifically involved at the recombination junctions. This recombination hotspot is located within the 'highly recombinogenic' region of the full-length CaMV RNA that has been shown to promote viral recombination in dicot plants. Clustering of plasmid recombination events in this highly recombinogenic region, even in the absence of viral enzymes and other cis-acting elements proves that the plant cellular machinery alone is sufficient to recognize and act on these viral sequences. Our data also show the similarity between mechanisms underlying junction formation in dicot and monocot plants transformed using different procedures.

Base Sequence↗
PubMed1995

Lagging-strand origins of the promiscuous plasmid pMV158: physical and functional characterization.

The streptococcal plasmid pMV158 replicates by a rolling circle mechanism, which involves the generation of single-stranded plasmid DNA intermediates. This plasmid has the unique feature of having two lagging-strand origins of replication. One of these origins, termed ssoU, is functional in Streptococcus pneumoniae and in Bacillus subtilis in an orientation-dependent manner. The other origin, ssoA, is only functional in the former host. RNA polymerase seems to be involved in the initiation of the conversion of single- to double-stranded plasmid DNA from both ssoA and ssoU. Mutational and deletion analyses have allowed us to define ssoA as being within a highly structured, non-coding 199 bp region. Within this region, two elements which are conserved in several rolling-circle replicating plasmids are located, the recombination site RSB and a 6 base consensus sequence. Both elements may play a role in the conversion of single- to double-stranded plasmid DNA.

Bacillus subtilis↗
PubMed1998

Molecular characterization of plasmid-mediated oxytetracycline resistance in Aeromonas salmonicida.

Using broth conjugation, we found that 19 of 29 (66%) oxytetracycline (OT)-resistant isolates of Aeromonas salmonicida transferred the OT resistance phenotype to Escherichia coli. The OT resistance phenotype was encoded by high-molecular-weight R-plasmids that were capable of transferring OT resistance to both environmental and clinical isolates of Aeromonas spp. The molecular basis for antibiotic resistance in OT-resistant isolates of A. salmonicida was determined. The OT resistance determinant from one plasmid (pASOT) of A. salmonicida was cloned and used in Southern blotting and hybridization experiments as a probe. The determinant was identified on a 5.4-kb EcoRI fragment on R-plasmids from the 19 OT-resistant isolates of A. salmonicida. Hybridization with plasmids encoding the five classes (classes A to E) of OT resistance determinants demonstrated that the OT resistance plasmids of the 19 A. salmonicida isolates carried the class A resistance determinant. Analysis of data generated from restriction enzyme digests showed that the OT resistance plasmids were not identical; three profiles were characterized, two of which showed a high degree of homology.

Abscess↗
PubMed2003

Nucleotide sequence based characterizations of two cryptic plasmids from the marine bacterium Ruegeria isolate PR1b.

Two plasmids, 76 and 148 kb in size, isolated from Ruegeria strain PR1b were entirely sequenced. These are the first plasmids to be characterized from this genus of marine bacteria. Sequence analysis revealed a biased distribution of function among the putative proteins encoded on the two plasmids. The smaller plasmid, designated pSD20, encodes a large number of putative proteins involved in polysaccharide biosynthesis and export. The larger plasmid, designated pSD25, primarily encodes putative proteins involved in the transport of small molecules and in DNA mobilization. Sequence analysis revealed uncommon potential replication systems on both plasmids. pSD25, the first repABC-type replicon isolated from the marine environment, actually contains two repABC-type replicons. pSD20 contains a complex replication region, including a replication origin and initiation protein similar to iteron-containing plasmids (such as pSW500 from the plant pathogen Erwinia stewartii) linked to putative RepA and RepB stabilization proteins of a repABC-type replicon and is highly homologous to a plasmid from the phototrophic bacterium Rhodobacter sphaeroides. Given the nature of the putative proteins encoded by both plasmids it is possible that these plasmids enhance the metabolic and physiological flexibility of the host bacterium, and thus its adaptation to the marine sediment environment.

ATP-Binding Cassette Transporters↗
PubMed1977

Characterization of a plasmid from Streptomyces coelicolor A3(2).

Covalently closed circular deoxyribonucleic acid (DNA) with a molecular weight of 20 X 10(6) was identified in strains of Streptomyces coelicolor A3(2) of various fertility types. Hybridization studies and digestion by various restriction endonucleases indicated that the circular DNAs (pSH1) were identical regardless of the fertility type (UF, IF, or NF) of the strain from which it was isolated. The pSH1 DNA was cleaved to many fragments by the endonucleases HincII, SmaI, and SalI and to three or four fragments by BamHI and PstI. Plasmid pSH1 carries single sites for each of the two restriction enzymes, EcoRI and HindIII. These sites are 7.6 X 10(6) daltons apart. Attempts to isolate the fertility factor SCP1 as covalently closed circular DNA were unsuccessful. These data suggest that the biochemically isolated plasmid pSH1 is not identical to the genetically characterized fertility factor SCP1, which has been identified in an autonomous state in IF-type strains and in an integrated state in NF-type strains.

Bacteriocins↗
PubMed1997

Isolation and characterization of a cryptic plasmid from Erwinia citreus ATCC 31623.

A multiple-copy plasmid pPZG500 (3.8 kb) was isolated from a phytopathogenic bacterium Erwinia citreus ATCC 31623. This is the smallest plasmid so far isolated from the genus Erwinia. The plasmid was partially characterized by a set of restriction enzymes and the unique restriction sites were mapped for HindIII, EcoRI, EcoRV and XBaI, while three sites were found for BglII. Nineteen other enzymes did not cut pPZG500. By deletion analyses minimal regions required for replication (ori) and segregational stability (par) were localized on 1.4 kb EcoRV/BglII and 0.7 kb Bgl/II/EcoRI fragments, respectively. The erythromycin resistance marker (Emr) was cloned into pPZG500 and two plasmid derivatives, pPZG502 and pPZG503, were constructed expressing erythromycin resistance as a good selective marker for recombinant selection in Erw. citreus and Escherichia coli. The segregational stability of both constructed plasmids during 90 generations in E. coli JM109 and Erw. citreus C-4 showed that plasmid pPZG503 lacking the presumptive par region was lost from the population at a higher rate. The results of this study demonstrate that plasmid pPZG500 and derivatives are suitable prerequisites for the construction of useful cloning vector(s) in the genus Erwinia.

DNA Replication↗
PubMed2004

Immunoliposomes carrying plasmid DNA: preparation and characterization.

The objective of this study was to characterize immunoliposomes carrying plasmid DNA with optimal encapsulation efficiency and antibody density. Plasmid DNA was encapsulated by the freezing/thawing method into liposomes composed of POPC (1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine), DDAB (didodecyl dimethyl ammonium bromide), DSPE-PEG 2000 (distearoyl phosphatidyl ethanolamine polyethylene glycol 2000) and DSPE-PEG 2000-maleimide. The liposomes carrying plasmid DNA were extruded through two stacked polycarbonate filters, of different pore size, to control the liposome size. Then, rat IgG molecules were conjugated to the liposomes. The immunoliposomes containing plasmid DNA were separated from the free plasmid DNA and unconjugated IgG by Sepharose CL-4B column chromatography. The DNA amount encapsulated was affected by DDAB (cationic lipid) concentration, the initial amount of plasmid DNA between 10 microg and 200 microg, the total lipid amount and plasmid DNA size, but not significantly by liposome size. By varying the ratio of DSPE-PEG 2000-maleimide to IgG, the number of IgG molecules per liposome was changed significantly.

Animals↗
PubMed2003

Study of recombinant micro-organism populations characterized by their plasmid content per cell using a segregated model.

Numerous observations from recombinant systems have shown that properties such as the specific cell growth rate and the plasmid-free cell formation rate are related, not only to the average plasmid content per cell, but also to the plasmid distribution within a population. The plasmid distribution in recombinant cultures can have an effect on the culture productivity that cannot be modelled using average values of the overall culture. The prediction of the behaviour of a plasmid content distribution and its causes and effects can only be studied using segregated models. A segregated model that describes populations of recombinant cells characterized by their plasmid content distribution has been developed. This model includes critical causes of recombinant culture instability such as the plasmid partition mechanism at cell division, plasmid replication kinetics and the effect of the plasmid content on the specific growth rate. The segregated model allows investigation of the effect of each of these causes and that of the plasmid content distribution on the observable behaviour of a recombinant culture. The effect of two partitioning mechanisms (Gaussian distribution and binomial distribution) on culture stability was investigated. The Gaussian distribution is slightly more stable. A small plasmid replication rate constant results in a very unstable culture even after short periods of time. This instability is dramatically improved for a larger value of this constant, hence improving protein synthesis. For a very narrow initial plasmid distribution, a given plasmid replication rate and partitioning mechanism can become broad even after a relatively short period of time. In contrast, a very "broad" initial distribution gave rise to a "Gamma-like" distribution profile. If we compare the results obtained in the simulations of the segregated model with those of the non-segregated one (average model), the latter model predicts much more stable behaviour, thus these average models cannot predict culture instability with the same precision. When compared with the experimental results, the segregated model was able to predict the practical behaviour with accuracy even in a system with a high plasmid content per cell and a high rate of plasmid-free cell formation which could not be achieved with a non-segregated model.

Journal Article↗
PubMed1990

Evaluation of restriction endonuclease fingerprinting of chromosomal DNA and plasmid profile analysis for characterization of multiresistant coagulase-negative staphylococci in bacteremic neonates.

A procedure was developed for restriction endonuclease fingerprinting (REF) of the chromosomal DNA of coagulase-negative staphylococci. A total of 48 isolates comprising 29 Staphylococcus epidermidis and 19 Staphylococcus haemolyticus isolates from blood and mucocutaneous sites of 15 premature neonates were characterized by REF, plasmid profile (PP) analysis, antimicrobial susceptibility testing, biotyping, and slime production. On the basis of REF analysis of chromosomal DNA, the 48 coagulase-negative staphylococcal isolates were subdivided into 10 subgroups, whereas PP analysis subdivided the strains into 20 distinct subgroups. REF analysis of total DNA (i.e., chromosome plus plasmid) resulted in the same 20 subgroups as were subdivided by PP analysis. The high discriminatory power of PP analysis was associated with the variability of plasmid content in coagulase-negative staphylococcal strains isolated during the outbreak. REF patterns were found to be stable both in vitro and in vivo. Isolates carried from 2 to 10 plasmids that ranged in molecular size from 0.9 to 39.5 megadaltons. Plasmids were disseminated among the coagulase-negative staphylococci, regardless of the genetic relatedness of their chromosomal DNAs. Hence, a lack of correlation existed between the grouping of isolates by REF analysis of chromosomal DNA and the grouping by PP analysis. There were one and two distinct chromosomal patterns among 4 of 4 blood cultures and 15 of 15 mucocutaneous cultures of S. haemolyticus, respectively. In contrast, a higher proportion of distinct chromosomal patterns was found for S. epidermidis in blood cultures (7 of 11 cultures) compared with those identified for isolates in mucocutaneous cultures (6 of 18 cultures). In summary, REF analysis of chromosomal DNA, rather than total DNA, is a useful marker for epidemiological investigations of coagulase-negative staphylococci. PP analysis can also be used to provide additional epidemiological information regarding the most recent genetic events.

Coagulase↗
PubMed1985

Large-scale preparation of ribulosebisphosphate carboxylase from a recombinant system in Escherichia coli characterized by extreme plasmid instability.

An ampicillin-resistant, RecA- strain of Escherichia coli (HB101) harboring the multicopy pBR322 plasmid containing the structural gene for ribulosebisphosphate carboxylase from Rhodospirillum rubrum was used to prepare large quantities of the carboxylase protein. This recombinant system was characterized by extreme plasmid instability, which resulted in part from the 1.7-fold faster growth rate of plasmid-free cells and in part from very rapid rates of plasmid segregation. The plasmid-containing organisms produced and excreted a large amount of beta-lactamase activity, with the result that ampicillin selection could only be maintained for a very short period of time, after which the plasmid-containing (carboxylase-producing) cells were overgrown by plasmid-free cells. The instability was so severe that even isolated colonies prepared on ampicillin-containing plates were impure and contained plasmid-free cells. Nevertheless, large quantities of carboxylase protein could be obtained from this system by using a highly dilute inoculum which allows selection of ampicillin-resistant (carboxylase-producing) organisms for a sufficient period of time so that the period of growth under nonselective conditions was minimized, and cells harvested at high cell densities contained large amounts of the carboxylase protein. In the present instance, 300-liter fermentations were initiated with a 0.3-microliter inoculum of freshly grown cells. After 20 h of growth in rich medium containing ampicillin, the harvested cells contained 74 g of ribulosebisphosphate carboxylase protein (average of two separate cultures). These results are discussed in terms of the general nature of plasmid instability and protocols available to minimize the effects of such instability.

Ampicillin↗
PubMed1995

Isolation of cryptic plasmids from moderately halophilic eubacteria of the genus Halomonas. Characterization of a small plasmid from H. elongata and its use for shuttle vector construction.

Three cryptic plasmids have been isolated from moderately halophilic eubacteria belonging to three species of the genus Halomonas. These three plasmids were designated pHE1 (4.2 kb, isolated from H. elongata ATCC 33174), pHI1 (48 kb, isolated from "H. israelensis" ATCC 43985), and pHS1 (ca. 70 kb, isolated from H. subglaciescola UQM 2927). Because of its small size, the plasmid pHE1 was selected for further characterization and construction of a shuttle vector for Halomonas strains. pHE1 was cloned into pBluescript KS and a detailed restriction map was constructed. Hybridization experiments excluded the existence of sequences homologous to pHE1 in total DNA from other strains of the genus Halomonas. Moreover, no DNA homology with pMH1, the only plasmid described so far from moderate halophiles, was found. Since pHE1 appeared to be unable to replicate in Escherichia coli cells, a number of mobilizable pHE1-derived hybrid plasmids were constructed that could be selected and maintained both in E. coli and in H. elongata. Finally, an improved shuttle vector, pHS15, was generated. The vector pHS15 contains an origin of replication from E. coli as well as one from H. elongata, a streptomycin resistance gene for positive selection in moderate halophiles, a number of unique restriction sites commonly used for cloning, and the mobilization functions of the broad host range IncP plasmid RK2. The vector pHS15 was readily mobilized by the RK2 derivative pRK2013 to all Halomonas strains tested so far. This is the first report on the development of a cloning vector useful for moderately halophilic eubacteria.

Genetic Vectors↗