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.     

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.     

Conjugative transfer of multiple antibiotic resistance markers in Streptococcus pneumoniae.

Two antibiotic-resistant isolates of Streptococcus pneumoniae were investigated for conjugative transfer of their drug resistance markers into streptococcal (groups B and D) and pneumococcal (encapsulated and non-encapsulataed) recipients. Of these, 7 wild-type donor pneumococci transferred all their resistance markers (except Pc [penicillin], Su [sulfonamide], and Tp [trimethoprim]) into group D Streptococcus and non-encapsulated S. pneumoniae recipients at a low frequency (10(-5) to 10(-6)). The resistance markers transferred were Tc (tetracycline); Tc and Cm (chloramphenicol); Tc and MLS (macrolides, lincosamides, and streptogramin B); Tc, MLS, Km (kanamycin), and Cm. The transconjugants obtained retransferred their resistance markers into appropriate streptococcal or pneumococcal recipients or both. The resistance markers of streptococcal transconjugants could not be cured by chemical agents. All attempts to detect extra-chromosomal deoxyribonucleic acid from pneumococcal or streptococcal transconjugants were unsuccessful. The molecular weight of a streptococcal conjugative R plasmid (pIP501) was investigated after transfer into the non-encapsulated S. pneumoniae recipient and was found to be similar to that of the wild-type group B Streptococcus host (20 x 10(6)).     

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.     

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.     

Characterization and transferability of Clostridium perfringens plasmids.

Two strains of Clostridium perfringens resistant to clindamycin (Cl), chloramphenicol (Cm), erythromycin (Em), and tetracycline (Tc) were isolated in France in 1974 and 1975. For one of these strains, curing experiments and molecular characterization of the extrachromosomal DNA clearly demonstrate the existence of two plasmids, plP401 (54 kilobases) and plP402 (63 kilobases), which, respectively, code for Tc Cm and Em Cl resistance. With mixed cultures, the Tc Cm plasmid is transferable to sensitive strains of C. perfringens; a segregation of these markers is frequently observed during mating experiments. In contrast, the transfer of the naturally occurring plasmid Em Cl does not occur at a significant rate. In performing transfer experiments in axenic mice, we obtained a Cl^r Em^r Tc^r transcipient whose chromosomal properties are those of a hybrid. When used in mating as a parental strain, this strain promotes chromosomal gene exchange. The role of the plasmid in this phenomenon is discussed, these transcipients being generally Cl^r Em^r Tc^r. The plasmid transfer is not limited to antibiotic resistance plasmids, the transferability of a bacteriocinogenic plasmid, plP404, harbored by C. perfringens BP6K-N5 being shown also. The transfer mechanism remains to be proved; it might be a conjugation process, a cell-to-cell contact being necessary for the transfer.     

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.     

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.     

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.     

Molecular and genetic studies of an R factor system consisting of independent transfer and drug resistance plasmids

Certain genetic, structural, and biochemical properties of a class 2 R-factor system consisting of the conjugally proficient transfer plasmid I and the naturally occurring non-conjugative tetracycline (Tc) resistance plasmid 219 are reported. I and 219 exist as separate plasmid deoxyribonucleic acid (DNA) species in both Escherichia coli and Salmonella panama, having molecular weights of 42 x 10(6) and 5.8 x 10(6), respectively. The buoyant densities of I and 219 are 1.702 and 1.710 g/cm(3), respectively, in neutral cesium chloride. Although the Tc resistance plasmid is not transmissible in a normal conjugal mating, it is mobilized in a three-component mating by plasmid I and by certain other conjugative plasmids of the fi(+) or fi(-) phenotype. Mobilization does not appear to involve intermolecular recombination between plasmids, and no covalent linkage of resistance markers and fertility functions is observed. Transformation of CaCl(2)-treated E. coli by plasmid DNA is shown to be a useful procedure for studying the biological properties of different plasmid molecular species that have been fractionated in vitro, and for selectively inserting non-self-transmissible plasmids into specific bacterial strains. The effects of tetracycline on the rate of protein synthesis carried out by plasmid 219 were studied by using isolated E. coli minicells into which this plasmid had segregated. Consistent with the results of earlier investigations showing the inducibility of plasmid-mediated Tc resistance in E. coli, the antibiotic was observed to stimulate protein synthesis in minicells carrying the plasmid 219 and totally inhibit (3)H-leucine incorporation by minicells lacking the Tc resistance marker. Five discrete polypeptide species were synthesized by minicells carrying plasmid 219; exposure of minicells or parent bacteria to Tc resulted in specific and reproducible changes in polypeptide synthesis patterns.     

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.     

Selective advantage of deletions enhancing chloramphenicol acetyltransferase gene expression in Streptococcus pneumoniae plasmids

A hybrid plasmid, pJS37, was made by combining pLS1, which confers tetracycline (Tc) resistance, and pC194, which confers chloramphenicol (Cm) resistance. Both pJS37 (7.3 kb) and its derivative pJS140 (6.0 kb), from which pC194 replication genes were removed, were structurally and segregationally stable when introduced into Streptococcus pneumoniae and grown either in the presence of Tc or in the absence of drug. However, both hybrid plasmids underwent systematic deletion when grown in the presence of Cm. One of the deleted forms, pJS4 (3.4 kb), could not be maintained in the absence of a helper plasmid; two others, pJS3 (4.1 kb) and pJS5 (3.8 kb), lost the tet gene but retained the replication functions of pLS1. They both expressed very high levels of Cm acetyltransferase (CAT), which, in the case of pJS5, were constitutive. Nucleotide sequence determination of the deletion junctions in pJS3 and pJS5 indicated that the deletions occurred, presumably by recombination, between short direct repeats of 6 and 9 bp, respectively. In both cases the tet promoter was juxtaposed to the cat gene. In the case of pJS5, the deletion removed a sequence that sequestered the ribosome-binding site (RBS) for cat, thereby rendering constitutive the production of CAT. The increased resistance to Cm afforded by the hyperexpression of the cat gene apparently provided a positive selective advantage for the accumulation of the deleted forms in the plasmid pool.     

Expression of chloramphenicol resistance specified by plasmid pHY416 hosted inCorynebacterium glutamicum

Chloramphenicol (Cm)-resistant colonies ofCorynebacterium glutamicum strain AS019 hosting plasmid pHY416 occurred at a frequency of 4×10^–7 when this strain, which normally expresses kanamycin (Km) resistance only, was placed under positive selection. These isolates produced a chloramphenicol acetyltransferase (CAT) and were tenfold more resistant to Cm than the parent strain. Resistance was lost in the absence of Cm selection but could be reselected from Km-resistant clones by reapplying Cm selection. Restriction endonuclease analyses of plasmids extracted from Cm-sensitive and-resistant strains indicated that expression of CAT activity corresponded to a loss of 0.9 kb of DNA from plasmid pHY416.     

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.     

Resistance plasmids in Pseudomonas cepacia 4G9.

Pseudomonas cepacia 4G9 utilizes 2-tridecanone as its sole carbon source and has been shown to be resistant to a variety of antibiotics. To ascertain whether any of these characteristics were plasmid mediated, Escherichia coli HB101 was transformed with plasmid DNA isolated from Pseudomonas cepacia 4G9. No 2-tridecanone-utilizing transformants were obtained. Tetracycline (Tc)- and ampicillin (Ap)- resistant transformants were obtained at a low frequency. Plasmid deoxyribonucleic acid from antibiotic-resistant E. coli HB101 transformants had molecular weights of 2.9 x 10(6) for pJW2 Tcr and 5.4 x 10(6) for pJW3 Apr as determined by electron microscopy. Electron microscopy of plasmid deoxyribonucleic acid from P. cepacia 4G9 revealed a single plasmid species, pJW1 of 1.78 x 10(6). Tetracycline resistance in both P. cepacia 4G9 and E. coli HB101(pJW2) was inducible, whereas ampicillin resistance in P. cepacia 4G9 was constitutive. The level of ampicillin resistance coded by pJW3 was lower in P. cepacia 4G9 than in the transformant E. coli HB101(pJW3).     

铜绿假单胞菌PA16株粘附性、菌毛与质粒关系的研究

为探讨PA的质粒与粘附性及质粒与菌毛的关系,围绕PA16株的耐药性与质粒的关系、质粒与菌毛及粘附性的关系作了一系列的研究,结果表明PA16对所测的7种抗生素全部耐药,其MIC＞400 mg/L;PA16仅含有一种27.3 kb(18 Mu)的质粒.转化后此质粒也使JM109获得了对四环素的耐药性.消除此质粒后,PA16对四环素的耐药性消失.粘附试验证明PA16质粒消除株对尿道上皮细胞的粘附能力较野生株显著性减小(P＜0.05),同时,透射电镜照片显示PA16野生株表面有致密、纤细刚直的菌毛,而PA16质粒消除株表面几乎无菌毛可见.     

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.     

Genetic and Biophysical Study of R Plasmids Conferring Sulfonamide Resistance in Shigella Strains Isolated in 1952 and 1956

The conjugative plasmids determining sulfonamide resistance in five Shigella strains, each isolated from a different patient, have been characterized. One S. flexneri 2a strain, isolated in 1952, harbored an fi(+) plasmid of molecular weight 53 x 10(6), which specified synthesis of F-like pili and bore determinants for sulfonamide resistance (Su) and bacteriocinogeny (Col). This plasmid was compatible with plasmids of groups F(I), F(II), I(alpha), and P. A second S. flexneri 2a strain isolated in 1952 harbored an fi(-) plasmid of molecular weight 59 x 10(6), bearing the Su determinant and compatible with all plasmids tested. This strain also harbored an fi(+) group-F(II) plasmid of molecular weight 42 x 10(6), which bore the Col determinant and specified synthesis of F-like pili. Three S. dysenteriae 2 strains isolated in 1956 carried apparently identical fi(-) plasmids of molecular weight 58 x 10(6), which bore the Su determinant, could form transconjugants in Pseudomonas but not in Proteus, and were incompatible with the P-group plasmid RP4.     

Plasmid-mediated chloramphenicol resistance in Staphylococcus hyicus

A small plasmid of 3.95 kb, encoding resistance to chloramphenicol (Cm) was detected in three of 33 Staphylococcus hyicus strains. The plasmid in each of the three strains was indistinguishable by Southern-blot hybridization and restriction enzyme analysis. It was shown by curing and by transformation to specify resistance to Cm. A preliminary restriction map of the plasmid, designated pSC2, is presented. Chloramphenicol acetyltransferase was demonstrated by enzyme assay and by SDS-PAGE of cell-free lysates of pSC2 transformants.     

Plasmids in Bacillus stearothermophilus coding for bacteriocinogeny and temperature resistance.

Obligately thermophilic strains of Bacillus stearothermophilus were screened for the presence of plasmids by agarose gel electrophoresis. All strains in our collection contained large plasmids (20 x 10(6)-80 x 10(6)) and were divided into four groups with respect to their plasmid pattern and production of bacteriocins. The major plasmid species were designated pSE407 (38.7 x 10(6)), pSE409 (29.0 x 10(6)), pSE411 (21.5 x 10(6)), and pSE410 (23.5 x 10(6)). Their physical endonuclease maps were constructed, and by Southern blots and hybridizations it was shown that these plasmids were related. From curing experiments and electrotransformations (electroporations) we conclude that pSE407, pSE410, and pSE411 code for temperature resistance. In addition pSE410 codes for bacteriocin production and resistance. Plasmid pSE409 probably also codes for bacteriocin production and resistance.