Experimental production of colicin type V ofShigella sonnei unusual in nature

Col V factor of Fredericq’s strainEscherichia coli 1.7a was mobilized for a transfer by the R (Sm, Cm, Tc, Su) factor ofShigella sonnei strain in three component test. The strainE. coli K13 HfrR (a universal indicator for evidence of colicinogeny) andS. sonnei strain became the recipient of both plasmids.S. sonnei, a producer of colicin V has not been found in natural conditions as yet.     

Characterization of the plasmids comprising the “R Factor” R5 and their relationships to other R plasmids

The “R factor” R5 confers resistance to tetracycline (Tc), streptomycin (Sm) and spectinomycin (Sp), chloramphenicol (Cm), sulfonamides (Su), kanamycin (Km), and mercuric ion (Mer). This phenotype is mediated by the presence of two R plasmids: pMH1 and pMH2, having approximate weights of 18.5 and 62 megadaltons (Mdal), respectively. pMH1 encodes Sm, Su, Cm, Mer, and Km resistance, and is nonconjugative. pMH2 confers Sm, Su, Cm, Mer, and Tc resistance, is conjugative, and belongs to the FII incompatibility group. NR79 is a 63-Mdal R plasmid encoding the same resistances as “R5,” and was derived from the same geographical source. It belongs to the FII incompatibility group and is conjugative. Analysis of restriction endonuclease digestion patterns and polynucleotide sequence homologies indicate that pMH1, pMH2, and NR79 are closely related. In addition, pMH2 and NR79 exhibit nearly complete homology to R100. Restriction endonuclease maps and resistance gene locations for pMH1, pMH2, and NR79 have been derived and a model for the evolutionary relationships of these plasmids is presented.     

Drug Resistance and Broad Geographical Distribution of Identical R Plasmids of Pasteurella piscicida Isolated from Cultured Yellowtail in Japan

An MIC test of 12 chemotherapeutic agents performed on 175 strains of Pasteurella piscicida collected from cultured yellowtail (Seriola quinqueradiata) in different areas of Japan from 1989 to 1991 revealed 152 strains (87%) with resistance to combinations of ampicillin (AP), chloramphenicol (CP), kanamycin (KM), nalidixic acid (NA), sulfamonomethoxine (SA), tetracycline (TC), and/or trimethoprim (TMP). The remaining 23 strains were sensitive to all the drugs tested: AP, cefazolin, CP, florfenicol (FF), furazolidone, KM, NA, novobiocin, SA, streptomycin, TC, and TMP. FF showed the most effective antibacterial activity against P. piscicida with MICs ranging from 0.004 to 0.6 μg/ml. One hundred and forty-nine of the 152 resistant strains carried transferable R plasmids encoding one of the Cp Km Sa Tc, Km Sa Tc, Km Sa, and Sa resistance. The most common resistance marker of transferable R plasmids identified in P. piscicida was Km Sa Tc. R plasmids encoding three different resistant markers were very similar on the basis of their digestion patterns with restriction endonucleases. There was homology among the DNAs of nine transferable R plasmids selected. Our findings suggest that multiple drug resistant strains of P. piscicida carrying transferable R plasmids with the same DNA structure are common in yellowtail farms and that the R plasmid has been retained within the P. piscicida population without change in their DNA structure according to geography and year.     

The R-factors of multiple antibiotic resistant faecal coliforms isolated from a domestic dog

The antibiotic resistant faecal flora of a domestic dog suffering from an acute enteric infection was examined. The flora exhibited overall resistance to a wide variety of antibiotics. However, following restoration of the animal to normal health, overall resistance to ampicillin (Ap), tetracycline (Tc), chloramphenicol (Cm) and streptomycin (Sm) was lost, although low numbers of bacteria resistant to these four antimicrobial agents could still be isolated up to one year later. A total of 11 strains were purified for further study. All 11 were positively identified as Escherichia coli and shown to be resistant to various combinations of the above antibiotics, and additionally to kanamycin (Km). Each strain harboured from one to five plasmids, although only four proved capable of transferring antibiotic resistance to Escherichia coli K-12. One of the strains was found to harbour two conjugal plasmids pNJ101 (60 Md) and pNJ102 (133 Md) which coded for resistance to Cm, Tc, Ap and Cm, Tc, Km respectively. A third plasmid pNJ103 (29 Md) remains cryptic. The possession of the two plasmids pNJ101 and pNJ102 appears to be an unstable situation as variants arose harbouring one or other of the plasmids.     

Molecular epidemiology of plasmid patterns in Shigella dysenteriae type I obtained from an outbreak in West Bengal (India)

Abstract Multiple antibiotic-resistant Shigella dysenteriae type 1 isolates from a recent epidemic in West Bengal (India) showed identical plasmid patterns. All isolates were resistant to ampicillin (Am), chloramphenicol (Cm), tetracycline (Tc), streptomycin (Sm) and trimethoprim (Tp) and contained 6 plasmids, ranging from 2.5–120 kb. The Am resistance determinant was located on the 120 kb plasmid. This plasmid was unstable when the S. dysenteriae strains were grown above 37°C. The Bangladesh strains of S. dysenteriae type 1 showed identical plasmid patterns, except that many isolates were Am-sensitive and lacked the 120 kb plasmid. In strains from both Bangladesh and West Bengal, predominantly group-B plasmids conferred resistance to Cm and Tc. Comparisons of Eco R1 fragments generated from the total plasmid DNA content of each strain support the view that the plasmids present in the S. dysenteriae type 1 strains isolated from all recent epidemics in India and Bangladesh were identical.     

Drug resistance of the causative agent of Grigo'rev-Shiga dysentery (Shigella dysenteriae 1)isolated in the USSR

Forty strains of S. dysenteriae 1 isolated in the USSR within 1986-1988 were tested for their resistance to 11 antibacterial drugs. It was shown that 92.5-97.5 per cent of the isolates were resistant to chloramphenicol (Cm) and tetracycline (Tc), 22.5 per cent to streptomycin (Sm), 17.5 per cent to nalidixic acid (Nal) and 10 per cent to ampicillin (Ap) and polymyxin (Pm). Resistance to Cm Tc (51.4 per cent) and Cm Tc Nal (13.5 per cent) represented the predominating phenotype. 35 per cent of the strains carried conjugative R plasmids. In the majority of the cases, the determinants of resistance to Cm and Tc were transferred, which must define the high frequency of the strains resistant to Cm and Tc. All the tested strains were sensitive to gentamicin, neomycin, rifampicin, cefamezin and ciprofloxacin. Since the strains of S. dysenteriae 1 proved to be highly sensitive to the tested drugs it appeared possible to consider them as the drugs of choice in etiotropic therapy of patients with dysentery caused by the pathogens of the Grigoryev-Shiga group.     

Characteristics of the pKMR plasmids found in Shigella flexneri

The clinical isolate of Sh. flexneri 1b, resistant to 5 antibiotics and sulfonamides, has been studied by the method of conjugation and found to have a group of transfer-suppressed pKMR-plasmids: pKMR 204-1 (Ap Sm Tc Cm Km Su), pKMR 204-2 (Sm Km Su), pKMR 204-5 (Km Su) and pKMR 204-7 (Sm Tc Cm Km Su), whose molecular weight was 99, 71.2, 73.8 and 59.5 Md respectively. The treatment of the plasmids with restriction endonuclease BamHI has revealed that plasmids pKMR 204-2 and pKMR 204-5 are definitely related to plasmid KMR 204-1, being its deletion mutants. At the same time plasmids pKMR 204-1 and pKMR 204-7 differ in their sensitivity to endonuclease BamHI and stably coexist within the same cell, thus seeming to belong to different compatibility groups.     

Deletion mutants from R plasmids with increased copy number.

Rms 201-12 and Rms 201-46 are R mutants with increased copy number, and are derived from a conjugative plasmid Rms 201 that encodes resistance to five drugs, ampicillin (Apc), tetracycline (Tc), chloramphenicol (Cm), streptomycin (Sm), and sulfonamides (Sa). The mutants expressed increased levels of resistance to Apc, Sm, and Cm, and a decreased level of resistance to Tc than those of the parent Rms 201 plasmid. When the Rms 201-12+ or Rms 201-46+ cells were inoculated onto plates containing a high concentration of Tc, colonies developed on the plate at a frequency of 10-3 to 10-4 after overnight incubation. The cells grown on the Tc plate carried a tet (Tc gene)-deleted R mutant besides tet-possessing Rms 201-12 or Rms 201-46, and we isolated the tet-deleted R mutant by purifying the R+ cells on drug-free plates. On the other hand, various deletion mutants possessing tet were isolated by prolonged culture of the cells. We have presented a circular gene order of Rms 201 by comparing the genetic markers of all deletion mutants derived from Rms 201, Rms 201-46, and Rms 201-12. The gene(s) regulating the copy number was closely linked to the rep gene. The gene(s) specifying entry exclusion was jointly lost with the tra region.     

Heteroduplex mapping of small plasmids derived from R-factor R12: in vivo recombination occurs at IS1 insertion sequences.

Small, autonomously replicating plasmids derived by in vivo recombination from R-factor R12 (= R100) have been structurally mapped by heteroduplex formation between the plasmids and an R-factor which is structurally closely related to R6-5. Recombination resulting in generation of the small resistance-free plasmids occurs between the (IS1)b insertion sequence and various other sites on the opposite side of an origin of replication. A larger R12-derived plasmid pSM17, carrying streptomycin (Sm), sulfadiazole (Sa), and chloramphenicol (Cm) resistances, has recombined in a similar manner but at the (IS1)a sequence. A new structural coordinate origin for R100 and for partially homologous R-factors is proposed based upon the location of the (IS1)b sequence.     

Characterization and construction of molecular cloning vehicles within Staphylococcus aureus.

Four chloramphenicol resistance (Cm) and four tetracycline resistance (Tc) plasmids from Staphylococcus aureus were characterized by restriction endonuclease mapping. All four Tc plasmids had molecular masses of 2.9 megadaltons (Mdaltons) and indistinguishable responses to seven different restriction endonucleases. The four Cm plasmids (pCW6, pCW7, pCW8, and pC221) had molecular masses of 2.6, 2.8, 1.9, and 2.9 Mdaltons, respectively. The four Cm plasmids also differed both in the level of resistance to Cm and in susceptibility to retriction endonucleases. Single restriction endonuclease sites contained within each plasmid included the following: in pCW6 for HindIII, XbaI, HpaII, and BstEII; in pCW7 for HindIII, BstEII, BglII, HaeIII, and HpaII; in pCW8 for HindIII, HaeIII, and HpaII; in pC221 for HindIII, BstEII, and EcoRI. The molecular cloning capabilities of pCW8 and pC221 were determined. Cm and erythromycin resistance (Em) recombinant plasmids pCW12, PCW13, and pCW14 were constructed and used to transform S. aureus 8325-4. A 2.8-Mdalton HindIII fragment from plasmid pI258 was found to encode Em resistance and contain single sites for the retriction endonucleases BglII, PstI, HaeIII, and HpaII. The largest EcoRI fragment (8 Mdaltons) from pI258 contained the HindIII fragment encoding Em resistance intact. Cloning of DNA into the BglII site of pCW14 did not alter Em resistance. Cloning of DNA into the HindIII site of pCW8 and the HindIII and EcoRI sites of pC221 did not disrupt either plasmid replication of Cm resistance.     

Interaction between R-factors and the host cells. I. Lethal interference between R-factor determined streptomycin resistance and streptomycin dependence in Proteus mirabilis

Summary Interaction between R-factor determined streptomycin (Sm) resistance (R-Sm) and chromosomally determined Sm dependence (str-d) has been studied using three different approaches by (i) attempts to transfer by conjugation the R-factor into Sm-dependent recipients, (ii) transduction of str-d alleles into R ⁺ recipients, and (iii) investigation of Sm-dependent revertants of a suppressed Sm-dependent mutant harboring an R-factor. Evidence is presented which strongly suggests a lethal interference between the function of R-Sm genes and str-d allele determined Sm dependence in Proteus mirabilis.Abbreviations used Sm streptomycin sulfate - Cm chloramphenicol - Su sulphathiacol - Tc oxytetracycline - str-s,-r,-d alleles of the str locus, determining Sm sensitivity, resistance, and dependence, respectively. Resistance genes carried by the R-genote are designed as R-Sm, R-Cm, R-Su, R-Tc.     

Co-Transduction of Plasmids Mediating Resistance to Tetracycline and Chloramphenicol in Staphylococcus aureus

Independent plasmids mediating resistance either to tetracycline (Tc) or chloramphenicol (Cm) were transduced successively into Staphylococcus aureus strain 8325. From this doubly resistant donor strain, Tc was co-transduced with a frequency of 40 to 50% when Cm was selected. Co-transduction of Cm was 5 to 10% with Tc selection. Plasmid elimination was infrequent and restricted to the Cm plasmid. A variant, doubly resistant strain gave 100% co-transduction of Tc and Cm and a high rate of joint elimination of both plasmid markers. Co-transduction of the plasmids from recombination-deficient donor strains was much reduced if the plasmids had been introduced separately into the donor strain, but occurred at the normal high rate if they had been introduced jointly. The plasmids were co-transformed at relatively low rates with closed circular deoxyribonucleic acid (DNA) from doubly resistant donors, but not with DNA from a mixed lysate of singly resistant strains. Our evidence favored a hypothesis of recombination-dependent, reversible linkage between the two plasmids as the basis for their co-transduction. Examination of plasmid DNA from the doubly resistant strains by ultracentrifugal and electron microscopic methods did not disclose any physical differences between singly and doubly resistant strains that might account for the observed co-transduction.     

Use of differential agar media for detection of cloned DNA fragments in the tetracycline and chloramphenicol resistance genes of pBR322

A method for detecting newly cloned DNA fragments in pBR322-based vectors was devised for use in DNA probe production. Escherichia coli strain DH5 containing plasmids with different resistance patterns to tetracycline (Tc) and chloramphenicol (Cm) were grown on nonpigmented media, blotted, transferred, and incubated for 2 h on MacConkey agar containing Tc or Cm. Resistant colonies changed color to pink as they began fermenting the lactose on the agar, while sensitive colonies remained white but were still viable and could be subcultured. This method can be applied to the detection of other plasmids with insertional inactivation of Tc or Cm resistance marker genes following successful cloning experiments, especially if pUC18 or M13 is not a possible vector. It eliminates 1 day of culture and the labor involved in individually transferring hundreds of colonies.     

Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis

Summary Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping. Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis. In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm. The genes are considered as chromosomal, since no association with endogenous plasmids was detectable. Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194. The subunit size of the clostridial CAT enzymes expressed in E. coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively. The C. butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.     

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.     

Transposon Tn5 mutagenesis of genes for the photosynthetic apparatus in Rhodopseudomonas capsulata

Summary Three plasmids containing the transposon Tn5, i.e. pSUP201::Tn5, pACYC184::Tn5 and pJB4JI were transferred from Escherichia coli to Rhodopseudomonas capsulata in order to mutagenize the genome. Mutants defective in bacteriochlorophyll and carotenoid synthesis and mutants unable to form the photochemical reaction center or one of the light-harvesting complexes were isolated. Of special interest were mutants that could not form the light-harvesting complex B800-850. Two of these mutants synthesized only two of the three polypeptides of this complex whereas the corresponding near infrared absorbance bands were not observed. Complementation analysis with the Rprime plasmid pRPS404, which contains a 50 kb region of the genome of R. capsulata carrying most genes responsible for expression of photosynthetic apparatus, revealed that some genes of the B800-850 light-harvesting complex lie outside this photosynthetic gene cluster.Abbreviations Bchl Bacteriochlorophyll - Cm chloramphenicol - Km kanamycin - Tc tetracycline - Ap ampicillin - Gm gentamicin - Spc spectinomycin     

Level and nature of the drug resistance of Shigella flexneri]

Characteristics of antibiotic resistance of 300 strains of Shigella flexner 2a isolated from patients within 1976--1977 in the regions where these bacteria were very rare for a long period of time were studied. It was shown that most of the isolates were resistant to chloramphenicol (88.1 per cent), tetracycline (94.8 per cent), streptomycin (89.1 per cent), polymyxin M (82.4 per cent) and others. 46.5--61.6 per cent of the isolates were resistant to neomycin antibiotics. A high level of the resistance was also noted: the bactericidal effect was registered in 52.0-74.0 per cent of the cultures at a dose of 500--1000 microgram/ml. 91.4 per cent of the strains possessed multiple dug resistance, 78.8 per cent of them being simultaneously resistant to 4--7 drugs. Transmissive R-plasmids were found in 68.8 per cent of the isolates. After exposure to acridine dyes the plasmid nature of the resistance was confirmed in 72.3 per cent of the cultures. Variability of the r-determinant sets in r-plasmids was noted. Strains (64.9 per cent) carrying r-determinants Tc, Cm, Sm and Tc, Cm were more frequent. Strains with one transmissive r-determinant were usually solitary.     

R-Plasmids in Pasteurella multocida.

Multiple drug-resistant strains of Pasteurella multocida were associated with a high incidence of fatal pneumonia in feedlot cattle. A representative strain, CAH160, resistant to tetracycline (Tc), streptomycin (Sm), and sulfonamide (Su) was studied. The minimal inhibitory concentration (MIC) of Tc was 32 μg/ml while Sm had an MIC of 256 μg/ml. Plasmid DNA was isolated from CAH160 by cesium chloride-ethidium bromide centrifugation. Agarose gel electrophoresis showed that at least three distinct species of plasmid DNA were present. DNA isolated from CAH160 was used to transform Escherichia coli K12 strain C600 r_k^?m_k^?. Transformants resistant to Tc; to Sm, Su; and to Tc, Sm, Su were obtained. Contour length measurements of plasmid DNA isolated from transformant cells showed that Tc resistance was associated with a 3-Mdal plasmid (pSR10), while Sm, Su resistance resided on a 2.7-Mdal molecule (pSR11). More than 20% of the transformants were resistant to Tc, Sm, Su and contained both plasmid species. In E. coli the MIC of Tc was 256 μg/ml and that of Sm was 64 μg/ml. The buoyant density of pSR10 was 1.699 g/cm³, while the density of pSR11 was 1.709 g/cm³.     

Translocation of sequences encoding antibiotic resistance from the chromosome to a receptor plasmid in Salmonella ordonez

Summary Salmonella ordonez strain BM2000 carries kanamycin (Km), ampicillin (Ap), spectinomycin (Sp), chloramphenicol (Cm), tetracyline (Tc), and sulfonamide (Su) resistance and production of colicin Ib (Cib). The Km and Cib characters were carried by a 97kb IncI1 plasmid (pIP565). In addition to the Km and Cib traits, all or part of the other antibiotic resistance (R) determinants could be transferred by conjugation from S. ordonez to Escherichia coli where all the acquired characters are borne by an IncI1 plasmid, designated complete or partial composite plasmid respectively. DNA from pIP565 and composite plasmids and total DNA from strain BM2000 were studied by agarose and polyacrylamide gel electrophoresis following digestion with restriction endonucleases, and by Southern hybridization. These comparative analyses enabled us a) to show that acquisition by pIP565 of resistance to all or some of the antibiotics was due to the insertion of a single DNA fragment into the receptor plasmid; b) to detect two types of composite plasmids with regard to the specificity of insertion into pIP565 and the mapping of the inserts; c) to demonstrate that the ApCmSpSuTc resistance determinants were integrated into S. ordonez BM2000 chromosomal DNA; d) to map the restriction fragments of the translocatable sequence integrated into strain BM2000 chromosome or into pIP565.The results obtained suggest that two distinct mechanisms for the translocation of the R determinants coexist in S. ordonez BM2000. Recombination between two of the four directly repeated copies of the IS-like sequence (IS1522) present in S. ordonez chromosome leads to the circularisation of all or part of the AmCmSpSuTc R determinants and is followed by either 1) a second recombination with the copy of IS1522 in pIP565 (Type I composite plasmids), or 2) transposition of precise groups of characters in various sites of pIP565 (Type II composite plasmids).     

Fecal coliforms in sewage. II. Transfer of markers and the presence of plasmids

The transference of the genetic markers and the presence of DNA plasmidial in 240 cultures of Escherichia coli was investigated. The strains were originated from Waste Treatment Plant (inffluent and effluents) located in Ilha do Governador, Rio de Janeiro. By conjugation analysis, E. coli K 12 allowed the isolation of the transconjugants resistant to antibiotics Su, Sm, Tc, Cm, Ap; heavy metals (Cu, Hg and Zn) and colicinogenic factors (Ia, Ib and V) mainly in coliforms isolated Cm and Ap from the terminals of the treatment plant. The percentual distribution of the plasmids was prevalent in the cultures of E. coli originated from material collected in the effluents and reached a rate higher than 65%.