Plasmids and transposable elements in Salmonella wien.

The plasmids from six clinical strains of Salmonella wien have been characterized. All the S. wien strains were found to carry three types of plasmids: an IncFI R-Tc Cm Km Ap (resistance to tetracycline, chloramphenicol, kanamycin, and ampicillin) plasmid, either conjugative or nonconjugative, of large size (90 to 100 megadaltons); an R-Ap Su Sm (resistance to ampicillin, sulfonamide, and streptomycin) plasmid of 9 megadaltons; and a very small (1.4 megadaltons) cryptic plasmid. The characteristics of conjugative R plasmids, recombinant between F'lac pro and the FI nonconjugative plasmid, indicated that regions coding for the donor phenotype were present on this plasmid. The molecular and genetic features of the R plasmids were very close to those described for the R plasmids isolated from S. wien strains of different origin. This fact supported the hypothesis of a clonal distribution of this serotype in Algeria and Europe. The analysis used to identify transposable elements showed the presence of only TnA elements, which were located on both the R-Tc Cm Km Ap and R-Ap Su Sm plasmids. They contained the structural gene for a TEM-type beta-lactamase and had translocation properties analogous to those reported for other TnA's.     

Cloning and characterization of EcoRI and HindIII restriction endonuclease-generated fragments of antibiotic resistance plasmids R6-5 and R6

Summary DNA fragments generated by the EcoRI or HindIII endonucleases from the low copy number antibiotic resistance plasmids R6 and R6-5 were separately cloned using the high copy number ColEl or pML21 plasmid vectors and the insertional inactivation procedure. The hybrid plasmids that were obtained were used to determine the location of the EcoRI and HindIII cleavage sites on the parent plasmid genomes by means of electron microscope heteroduplex analysis and agarose gel electrophoresis. Ultracentrifugation of the cloned fragments in caesium chloride gradients localized the high buoyant density regions of R6-5 to fragments that carry the genes for resistance to streptomycin-spectinomycin, sulfonamide, and mercury and a low buoyant density region to fragments that carry the tetracycline resistance determinant. Functional analysis of hybrid plasmids localized a number of plasmid properties such as resistances to antibiotics and mercury and several replication functions to specific regions of the R6-5 genome. Precise localisation of the genes for resistance to chloramphenicol, kanamycin, fusidic acid and tetracycline was possible due to the presence of identified restriction endonuclease cleavage sites within these determinants.Only one region competent for autonomous replication was identified on the R6-5 plasmid genome and this was localized to EcoRI fragment 2 and HindIII fragment 1. However, two additional regions of replication activity designated RepB and RepC, themselves incapable of autonomous replication but capable of supporting replication of a linked ColE1 plasmid in polA^– bacteria, were also identified.     

Characterization of pRAS1-like plasmids from atypical North American psychrophilic Aeromonas salmonicida

Atypical psychrophilic Aeromonas salmonicida isolates were obtained from farmed and wild fish in Northeastern North America. These bacteria were isolated between 1992 and 2001 and carried tetracycline resistance (Tc(r)) plasmids of approximately 58 kb. The nine isolates had plasmids which could be divided into four groups based on the specific tetracycline resistance (tet) gene carried [tet(A) or tet(B)], incompatibility of the plasmid [IncU or other], whether the plasmid carried the IS6100 sequences, the sul1 gene, coding for sulfonamide resistance, the dfrA16 gene, coding for trimethoprim resistance, and/or carried a complete Tn1721, and their ability to transfer their Tc(r) plasmids to an Escherichia coli recipient at 15 degrees C. Five of the isolates, with genetically related Tc(r) plasmids, were able to transfer their plasmids to an E. coli recipient at frequencies ranging from 5.7x10(-4) to 2.8x10(-6) per recipient. The 1992 isolate carried a genetically distinct plasmid, which transferred at a slightly higher rate. The three remaining isolates carried one of two genetically different plasmids, which were unable to transfer to an E. coli recipient. Conjugal transfer at 15 degrees C is the lowest temperature that has been documented in bacteria.     

淋病奈瑟菌粘附性质粒的研究

从1株临床分离的淋病奈瑟菌(N.gonorrheae,Ng)分别检测出耐氨苄青霉素(AMPr)及四环素(TCr)的2种质粒(R质粒),其分子量分别为25.2 Mu和4.5 Mu.进一步研究发现,编码TCr的质粒也编码普通菌毛,因而与其粘附性有关.消除此R质粒不但细菌的TCr消失,电镜下观察其菌毛几乎全部消失,其粘附性也显著降低,故此R质粒也被确认为是粘附性质粒(Adh质粒).     

Conjugative R-plasmid resistance of the causative agents of Yersinia infection

Nature, structure, occurrence and drug resistance of 160 strains of Y. pseudotuberculosis and 60 strains of Y. enterocolitica isolated from various sources within 1986-1988 were studied. In the strains of Y. pseudotuberculosis, the cell composition with respect to the requirements in calcium ions as well as the plasmid profiles with determination of the molecular weights of the plasmids in the antibiotic sensitive and resistant pathogens and R(+)-transconjugants were investigated. Some molecular genetic properties of the Yersinia R plasmids were also investigated. Antibiotic polyresistant strains of Y. enterocolitica were the most frequent donors of the R plasmids while the strains of Y. pseudotuberculosis were less frequently the donors, in the resistance pattern of which there were more frequent streptomycin and tetracycline resistance determinants. The conjugative R plasmids of Y. pseudotuberculosis were characterized by strict control of replication, repressed frequency of transfers, and a molecular weight of about 47 MD. Their replicones as a rule contained streptomycin and tetracycline markers determining resistance to streptomycin and tetracycline at the levels of 1250 and 156 micrograms/ml, respectively.     

Conjugative R plasmids isolated from hospital strains of Pseudomonas aeruginosa]

It was shown that Pseudomonas aeruginosa hospital strains isolated from patients and environment in the Republican Centre of Burns in Tbilisi contained conjugative R plasmids. The plasmids were marked pM15 and pM19, respectively. The plasmid pM15 determined resistance to carbenicillin, kanamycin and tetracycline and plasmid pM19 determined resistance to carbenicillin, kanamycin, tetracycline, chloramphenicol, gentamicin and streptomycin. Plasmid pM15 had a molecular weight of 45.8 MD and seven sites for EcoRI, six sites for HindIII and five sites for Hpa-I-restrictase. This plasmid, as others, belongs to the Inc-P1 incompatibility group.     

Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids

Ten plasmids were isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant thermophilic bacteria. Of the 10 plasmids tested, 2 could transform Bacillus subtilis, yielding resistance to specific antibiotics. Plasmid pTB20 (2.8 X 10(6) daltons, approximately 24 copies per chromosome) specifies resistance to tetracycline (Tcr), whereas pTB19 (17.2 X 10(6) daltons, approximately 1 copy per chromosome) renders the host resistant to both kanamycin and tetracycline (KMrTcr). Three plasmids were not self-transmissible. The restriction endonuclease cleavage maps of the two plasmids, pTB19 and pTB20, were constructed. pTB19 and pTB20, both of which were originally isolated from thermophilic bacilli, were tested for stability in B. subtilis. Digestion of pTB19 followed by ligation yielded deletion plasmids pTB512 (Kmr), pTB52 (Tcr), and pTB53 (KmrTcr). Determinants of Kmr, Tcr, and DNA replication were associated with EcoRI fragments R1b (4.2 X 10(6) daltons), R3 (2.8 X 10(6) daltons), and R1a (4.2 X 10(6) daltons), respectively. Restriction endonuclease cleavage maps of pTB51, pTB52, and pTB53 were constructed. Tetracycline resistance of pTB20 was confirmed to be in the EcoRI fragment (1.85 X 10(6) daltons).     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group.

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Enterococcus faecalis hemolysin-bacteriocin plasmids belong to the same incompatibility group

Plasmid pair coexistence was studied both among nine Enterococcus faecalis hemolysin-bacteriocin (Hly-Bcn) plasmids, including pJH2, pAD1, pAM gamma 1, and pIP964, and between pIP964 and five R plasmids. Some of the Hly-Bcn plasmids used were derivatives encoding resistance to erythromycin or tetracycline. The Hly-Bcn plasmids were incompatible with each other; 40 to 100% displacement was observed bilaterally for eight pairs and unilaterally for one pair. In contrast, pIP964 stably coexisted with each of the R plasmids. Entry exclusion was associated with incompatibility for most of the Hly-Bcn plasmids. The nine Hly-Bcn plasmids harbored by E. faecalis form a distinct incompatibility (Inc) group, designated IncHly.     

Involvement of plasmids in determining bacteriophage sensitivity in Salmonella typhimurium: genetic and physical analysis of phagovar 204

Strains resistant to the action of sulfa drugs and tetracycline were predominant among the antibiotic-resistant Salmonella typhimurium phagovar 204 isolated in Canada. Plasmid DNA was detected in cellular extracts of all strains examined. A number of these plasmids could be placed in specific incompatibility and size classes. Both resistance coding and cryptic plasmids were involved in determining phagovar 204. In one instance, phagovar 204 was derived from phagovar 36 in a two-step conjugation involving independent sulfadiazine and tetracycline resistance plasmids. In another, phagovar 204 was derived directly from phagovar 49 through the introduction of a single tetracycline-streptomycin R plasmid. The phagovar-determining plasmids ranged in size from 3.4 to 72 megadaltons.     

Generation of miniplasmids from copy number mutants of the R plasmid NR1.

Small, closed circular deoxyribonucleic acid molecules, called miniplasmids, were observed in Escherichia coli harboring copy number mutants of the R plasmid NR1 after growth in medium containing tetracycline. The level of tetracycline resistance conferred by the copy mutant plasmids was lower (3 to 6 microgram/ml) than that conferred by NR1 (100 MICROGRAM/ML). The presence of the miniplasmid enhanced the level of tetracycline resistance conferred by the copy mutant. Miniplasmids of molecular weights 4 X 10(6) to 13 X 10(6) were found. They carried no antibiotic resistance markers and could be eliminated by growth in the presence of chloramphenicol and/or streptomycin-spectinomycin. Studies with the restriction endonucleases EcoRI and Sal I indicated that the miniplasmids are derived from the region of the copy mutant plasmids that contains the origin for replication of the resistance transfer factor. There were approximately 12 copies of the miniplasmid per chromosome, compared with 3 and 6 copies of the copy mutants of NR1. The miniplasmids appeared to be incompatible with the copy mutant plasmids.     

Tetracyclines and Tetracycline Resistance in Agricultural Soils: Microcosm and Field Studies

The influence of the use of antibiotics on the prevalence of resistance genes in the environment is still poorly understood. We studied the diversity of tetracycline and sulfonamide resistance genes as influenced by fertilization with pig manure in soil microcosms and at two field locations. Manure contained a high diversity of resistance genes, regardless of whether it stemmed from a farm operation with low or regular use of antibiotics. In the microcosm soils, the influence of fertilization with manure was clearly shown by an increase in the number of resistance genes in the soil after manuring. Spiking of the tetracycline compounds to the microcosms had only little additional impact on the diversity of resistance genes. Overall, the tetracycline resistance genes tet(T), tet(W), and tet(Z) were ubiquitous in soil and pig slurries, whereas tet(Y), tet(S), tet(C), tet(Q), and tet(H) were introduced to the microcosm soil by manuring. The diversity of tetracycline and sulfonamide [sul(1), sul(2), and sul(3)] resistance genes on a Swiss pasture was very high even before slurry amendment, although manure from intensive farming had not been applied in the previous years. The additional effect of manuring was small, with the tetracycline and sulfonamide resistance diversity staying at high levels for the complete growth season. At an agricultural field site in Germany, the diversity of tetracycline and sulfonamide resistance genes was considerably lower, possibly reflecting regional differences in gene diversity. This study shows that there is a considerable pool of resistance genes in soils. Although it is not possible to conclude whether this diversity is caused by the global spread of resistance genes after 50 years of tetracycline use or is due to the natural background in soil resistance genes, it highlights a role that environmental reservoirs might play in resistance gene capture.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.     

Heterogeneity of tetracycline resistance determinants

We have found that tetracycline resistance on different naturally occurring bacterial plasmids is encoded by more than one genetic determinant. Using restriction enzyme analyses and DNA-DNA hybridization to specific ³²P-labeled genetic probes, we can define at least four genetically distinct tetracycline resistance determiants: Class A (the determinant on prototype plasmid RP1), Class B (that on R222), and Class C (that on plasmid pSC101). At least one other determinant, encoded by plasmid RA1, belongs to none of these three groups and has been designated Class D. These genetic classes confirm phenotypic differences in expression of resistance to tetracycline and tetracycline analogs encoded by the different plasmids.     

Incidence of the H2 group of plasmids in chloramphenicol-sensitive salmonella isolated in 1974 from clinical sources in Ontario.

Chloramphenicol resistance in salmonella obtained from clinical sources in Ontario was previously found to be often mediated by R plasmids of the H2 incompatibility group. In the present study 40 salmonella strains resistant to one or more of kanamycin, streptomycin, sulfonamides, or tetracycline but sensitive to chloramphenicol, were investigated to determine if they contained R plasmids. Self-transmissible plasmids were isolated from 17 of the strains, and 7 of those showed the bacteriophage inhibition and thermosensitive mechanism of transfer characteristic of H2 plasmids. Entry exclusion and incompatibility experiments confiremd their classification. The results demonstrate that in this population of salmonella, R plasmids of the H2 group are prevalent. Experiments with plasmid-specific phages indicate that the plasmids of this sample, which are not in the H2 group, do not belong to any of the F, I, N, P, or W incompatibility groups.     

Changes in the nature of the sensitivity of an E. coli M strain carrying Inc-group R plasmids, to coliphages T3 and T7

After the transfer of prototype plasmids R6K (IncX), R387 (IncK), R27 (IncH1) and T (IncN) to E. coli M nalr the appearance of histidine-dependent mutants (R27, T), histidine-leucine-dependent mutants (R6K), methionine-proline-dependent mutants (R387) was observed among the resulting transconjugates. The mutations of E. coli M nalr R+ cells induced by the introduction of the plasmids were accompanied by the transformation of the cells from the S-form into the R-form. In contrast to the prototrophs E. coli M nalr, the auxotrophs carrying plasmids R6K, R27, T acquired sensitivity to phage T7, and the methionine-proline-dependent mutant became sensitive to phages T and T7. The above-mentioned plasmids rendered E. coli M cells capable of synthetizing the donor pili. But the adsorption of phages T3 and T7 on the auxotrophic cells, both with and without plasmids, occurred due to their interaction with the cell-wall receptors.     

R factors of pathogenic strains of Escherichia coli isolated from infant diarrhoea

Several drug resistance patterns were determined in 170 pathogenic strains of E. coli isolated in 6 Polish towns from infant diarrhoea. The most frequent were strains resistant to 5 different drugs: ampicillin, tetracycline, chloramphenicol, streptomycin and sulfonamide. Conjugative R factors of 30 strains of the same resistance pattern (Ap Tc Cm Sm Su) were characterised by determining their Fi(F) character, incompatibility and molecular weight.     

Properties of an R Factor from Pseudomonas aeruginosa

An R factor from Pseudomonas aeruginosa, which confers resistance to penicillins, kanamycin, and tetracycline, was studied in Escherichia coli K-12. The R factor could coexist with F-like or I-like plasmids and therefore constituted a novel compatibility group. The R factor was transferable from E. coli to bacterial genera outside the Enterobacteriaceae (Pseudomonas and members of the Rhizobiaceae) to which transfer of F-like and I-like plasmids could not be demonstrated.     

Transformation of Pseudomonas aeruginosa and Escherichia coli with resistance plasmid DNA: formation of smaller conjugative plasmids from RPI.

Summary Sheared DNA from RPI, an R plasmid from a strain of Pseudomonas aeruginosa, was used to transform other strains of P. aeruginosa and Escherichia coli. From transformed cells other plasmids like RPI were isolated. These deletion plasmids were conjugally transferrable and confer resistance mainly against carbenicillin and tetracycline.     

Cointegrate formation between homologous plasmids in Escherichia coli.

Conjugation experiments were performed in which the donor was Escherichia coli K-12 strain KP245 containing either R plasmid NR1 plus an ampicillin-resistant derivative of ColE1 (*ColE1::Tn3, called RSF2124) or NR1 plus RSF2124 carrying a cloned EcoRI fragment of NR1. The recipient was the polA amber mutant JG112, in which RSF2124 cannot replicate. Ampicillin-resistant transconjugants can arise only when the genes for ampicillin resistance are linked to NR1 or are transposed to the host chromosome. When EcoRI fragment A of NR1 (20.5 kilobases) was cloned to RSF2124, the frequency of cotransfer of ampicillin resistance with tetracycline resistance was 25 to 60%. Plasmid DNA from these ampicillin-resistant transconjugant cells was analyzed by gel electrophoresis and was shown to be a cointegrate of NR1 and the RSF2124 derivative. Analysis of plasmid DNA isolated from donor cultures showed that the cointegrates were present before conjugation, which indicates that the mating does not stimulate cointegrate formation. When the cloned fragment was EcoRI fragment H of NR1 (4.8 kilobases), the frequency of cotransfer of ampicillin resistance with tetracycline resistance was about 4%, and the majority of the ampicillin-resistant transconjugants were found to contain cointegrate plasmids. When the donor contained NR1 and RSF2124, the frequency of cotransfer of ampicillin resistance was less than 0.1%, and analysis of plasmid DNA from the ampicillin-resistant transconjugants showed that Tn3 had been transposed onto NR1. These data suggest that plasmids which share homology may exist in cointegrate form to a high degree within a host cell.