Plasmids Controlling Synthesis of Hemolysin in Escherichia coli: Molecular Properties

Covalently closed extrachromosomal deoxyribonucleic acid (DNA) was isolated from alpha-hemolytic wild-type strains of Escherichia coli. Most strains examined were able to transfer the hemolytic property with varying frequencies to nonhemolytic recipient strains. Out of eight naturally isolated alphahemolytic E. coli strains, four contained a set of three different supercoiled DNAs with sedimentation coefficients of 76S (plasmid A), 63S (plasmid B), and 55S (plasmid C). The sedimentation coefficients and the contour lengths of the isolated molecules correspond to molecular weights of 65 x 10(6), 41 x 10(6), and 32 x 10(6). Three alpha-hemolytic wild-type strains carried only one plasmid with a molecular weight of 41 x 10(6), and one strain harbored two plasmids with molecular weights of 41 x 10(6) and 32 x 10(6). Alpha-hemolytic transconjugants were obtained by conjugation of E. coli K-12 with the hemolytic wild-type strains. A detailed examination revealed that plasmids with the same sizes as plasmids B and C of the wild-type strains can be transferred separately or together to the recipients. Both plasmids possess the hemolytic determinant and transfer properties. Plasmid A appears to be, at least in one wild-type strain, an additional transfer factor without a hemolytic determinant. In one case a hemolytic factor was isolated, after conjugation, that is larger in size than plasmid A and appears to be a recombinant of both plasmids B and C.     

Analysis of plasmid profile of antibiotic-resistant Enterobacteriaceae circulating in hospitals

Certain pheno- and genotype properties of S. typhimurium and some other representatives of Enterobacteriaceae resistant to antimicrobial drugs were studied. The strains were isolated from children with salmonellosis within 4 months when an infection hospital was subjected to microbiological observation. It was shown that by their antibiotic resistance, phagovars and molecular weights of the plasmid DNas, the strains S. typhimurium were similar to those isolated during hospital infections. The conjugative plasmids responsible for antibiotic resistance in some strains did not differ in their molecular weights and antibiotic resistance markers. The strains S. typhimurium similar in their pheno- and genotype properties were isolated only from 2 patients which allowed one to consider it possible that the patients were infected by the strains of common genesis. Analysis of nonpathogenic representatives of Enterobacteriaceae isolated from the patients along with the S. typhimurium strains confirmed the fact that the patients were infected with the same pathogenic strain.     

Molecular analyses of conjugative, gentamicin-resistance plasmids from staphylococcal clinical isolates

Gentamicin-resistant Staphylococcus aureus and Staphylococcus epidermidis strains which were isolated from infants with staphylococcal bacteremia were analyzed for the presence of self-transmissible gentamicin-resistance (Gmr) plasmids. Conjugative GMr plasmids of approximately 43.8-63 kilobases (kb) were found in all S. aureus strains. Inter- and intra-species transfer of Gmr plasmids by conjugation was observed from S. aureus to S. aureus and to S. epidermidis recipient strains. However, neither inter- nor intra-species transfer of gentamicin resistance by conjugation was observed with nine out of nine S. epidermidis donor strains which were mated with either S. epidermidis or S. aureus recipient strains. These conjugative Gmr plasmids were unable to comobilize a smaller (15-kb) plasmid present in all but two S. aureus clinical isolates. Many of the conjugative Gmr plasmids also carried genetic determinants for kanamycin, tobramycin, neomycin, and ethidium bromide resistance, and for beta-lactamase synthesis. EcoRI restriction endonuclease digests of the S. aureus Gmr conjugative plasmids revealed three different digestion patterns. Four EcoRI restriction endonuclease digestion fragments of 15, 11.4, 6.3, and 4.6 kb in size were common to all plasmids. These plasmids and conjugative Gmr staphylococcal plasmids from other geographical regions shared restriction digestion fragments of similar molecular weights. DNA hybridization with biotinylated S. aureus plasmid pIZ7814 DNA revealed a high degree of homology among these plasmids. A 50.9-kb plasmid from one of the nonconjugative S. epidermidis clinical isolates showed homology with the probe DNA but lacked a portion of a 6.3-kb fragment which was present in all conjugative plasmids and believed to carry much genetic information for conjugation.     

Plasmids controlling biodegradation of epsilon-caprolactam

Bacterial strains (190) capable of growth on epsilon-caprolactam as a sole source of carbon and nitrogen were isolated from epsilon-caprolactam industrial sewage. Most of the strains (90%) were found to contain plasmids. Some of the strains (36.8%) assigned provisionally to the genus Pseudomonas contained plasmids controlling epsilon-caprolactam catabolism. The plasmids had a molecular mass from 50 to 300 MDa. Certain plasmids differed in the frequency of conjugation transfer and in the presence of other genetic determinants (resistance against heavy metal ions) and also determined the different character of bacterial growth on epsilon-caprolactam and on its intermediate catabolites.     

Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains

Plasmid analysis of over 120 strains of Clostridium perfringens , isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6·2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.     

Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains

Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.     

Persistence and spread of a chloramphenicol resistance-mediating plasmid in antigenic types of Escherichia coli, pathogenic for piglets

All chloramphenicol-resistant Escherichia coli strains isolated from piglets in the State veterinary Serum Laboratory, Copenhagen, in 1974-1975 harbored plasmids of IncFII group with largely the same resistance markers. Two strains from 1978 carried plasmids with similar characters. Restriction enzyme analysis of DNA from these plasmids with restriction endonucleases EcoRI, BglII, and PstI shows that the Cm plasmids are extremely closely related; but the patterns obtained (particularly from PstI digests) enable the classification of the plasmids into groups. These bear a strong relation to time and place of isolation so that plasmids isolated on the same farm belong to the same group even when their host strains are of different antigenic types. It is concluded that these plasmids have evolved from a single plasmid.     

Occurrence of megaplasmids in halobacteria

Sixty-five halobacteria, including culture collection and freshly isolated strains from widely differing geographical areas, were examined for the presence of high molecular weight plasmids by agarose gel electrophoresis. Seventy-five per cent of all the strains were shown to harbour at least one plasmid. In the majority of strains three or four megaplasmids were detected. Approximate molecular weights of the plasmids were in the range < 100 to 300 megadaltons (Mdal). In most culture collection strains, two or three plasmids were demonstrated, except in two in which no plasmid was detected, and in two Haloarcula strains which were found to contain five and eight plasmids; four and six of the latter were more than 100 Mdal. No relationship between the plasmid profile of each strain and its taxonomic assignation nor its isolation source was found. Evidence is presented for the first time on the occurrence of megaplasmids in halobacteria.     

Plasmids mediating multiple drug resistance in group B streptococcus: transferability and molecular properties.

Group B β-hemolytic streptococci isolated from a number of geographic locations were investigated with regard to the genetic basis of their drug-resistance determinants. Strains have been identified carrying plasmid-mediated resistance to erythromycin, lincomycin, and streptogramin B (MLS resistance). These MLS-resistance plasmids all have molecular weights of approximately 17 × 10⁶ and are capable of transfer during mixed incubation by a mechanism that appears similar to conjugation. These plasmids were transferable to group B, D, F, and H recipients. Transfer could also be shown from groups F and H to group D recipients. Plasmids DNA isolated from recipient strains following transfer was identical with that of the donor. The similarity of restriction fingerprints of MLS-resistance plasmids from group A, B, and D isolates obtained suggest that they constitute a family of related plasmids.     

Distribution of citrate utilization plasmids in Salmonella strains of bovine origin in Japan.

Attempts to detect transferable citrate-utilizing (Cit) ability in enterobacterial strains were carried out by conjugation experiments. Of 318 strains of Salmonella typhimurium and 1 strain of Salmonella bredeney isolated from cattle in Japan from 1970 to 1979, 107 (33.5%) strains contained transferable Cit characters. Most of the strains transferred the Cit characters to recipient Escherichia coli more efficiently at 28 degrees C than at 37 degrees C, indicating that their transfer of the Cit character is thermosensitive. Transferred Cit characters were found in association with drug resistance markers such as ampicillin, chloramphenicol, kanamycin, streptomycin, sulfonamides, and tetracycline or with mercury resistance, but Cit plasmids conferring Cit ability alone were also obtained. Of 221 conjugative Cit plasmids tested for fertility inhibition (Fi), all but 2 were Fi- and exhibited thermosensitive transfer; 2 Cit plasmids showing the Fi+ character were also isolated from 2 S. typhimurium strains. No transferable Cit character was detected from strains of Proteus, Serratia, Klebsiella, Enterobacter, and Citrobacter spp. isolated from humans or cows in the present study. The utilization of tricarboxylic acids by strains with plasmid-borne Cit ability was examined, and two different patterns of utilization were found in the Cit+ E. coli transconjugants.     

Restriction enzyme digestion patterns ofFrankia plasmids

Summary After the initial screening of more than 200Frankia strains, the plasmid DNA observed in eight Frankiae was analyzed.In situ lysis was performed to obtain an estimate of their copy number and molecular weight. Four plasmid classes were distinguished, 7–9, 18–20, 30–35 and 50–55 kb. Twelve plasmids were thus analysed with restriction enzymes to determine their plasmid restriction patterns.While someFrankia plasmids with comparable molecular weights were found to be heterologous in their restriction enzyme pattern, an 8 kb plasmid found in bothFrankia sp. ArI3, isolated fromAlnus rubra andFrankia sp. CpI1 isolated fromComptonia peregrina showed undistinguishable fingerprints. Furthermore, an 18 kb plasmid found in the same two strains, also showed homologous restriction enzyme patterns. However, the copy numbers of the two ArI3 plasmids were higher than those of the CpI1 plasmids.Similarly, strains ACN1^AG, , isolated fromAlnus crispa all contained a 50 kb plasmid, and the three plasmids were found upon restriction analysis to be undistinguishable.In one strain, ARgX17c isolated fromAlnus rugosa, it was found through restriction enzyme analysis that two plasmids of a similar molecular weight were in fact heterologous.The possible origin of the homologous plasmids and their potential as specificFrankia markers to be used in ecological studies are discussed.     

Properties of Serratia marcescens strains isolated in septic diseases of newborn infants

As the result of the study of blood and liquor samples from 120 newborns, Serratia marcescens was isolated in 21 cases (17.5 %). 8 strains were isolated from the environment of these patients. Almost all strains isolated from both the patients and the environment (with the exception of one environmental strain) belonged to serotype 04. The isolated S. marcescens strains were resistant to penicillin, ampicillin, streptomycin, kanamycin, oxacillin, methicillin, ceporin and moderately sensitive to polymixin. 2 strains from the environment and 9 strains from the patients were mildly sensitive to gentamicin. In one hospital all isolated strains were found to have 2 transmissive R plasmids with the molecular weight 40 and 60 megadaltons. The presence of R plasmids with the same molecular weight in all S. marcescens strains isolated in this hospital, as well as their serological identity, suggest that in all patients infection originated from a common source.     

R plasmids among Gram-negative bacteria with multiple drug resistance isolated in a general hospital.

The incidence of conjugative R plasmids in multiple drug-resistant strains of gram-negative bacteria isolated in 1973 from patients in a 700-bed general hospital in Tokyo and some properties of the R plasmids isolated are described. Conjugative R plasmids were found in 52 of the 96 strains (54%), from which 74 R plasmids were demonstrated. It is remarkable that the isolation frequency of R plasmids mediating quadruple- or five-drug resistance was rather low, and the complete pattern of multiple resistance of the original isolates was only rarely transferred by conjugation. These results revealed the existing state of the distribution of R plasmids among hospital strains with multiple drug-resistance.     

Involvement of a Polyethylene glycol (PEG)-oxidizing Enzyme in the Bacterial Metabolism of PEG

Eighty-seven strains of acetic acid bacteria were surveyed for plasmids by CsCl-ethidium bromide equilibrium centrifugation of cell lysates. Twenty-seven of the 33 strains of Acetobacter were found to harbor plasmid DNA and most strains contained multiple species of low-molecular-weight plasmids. On the other hand, plasmid DNAs were detected in 23 of the 36 strains of Gluconobacter and most of them had molecular weights of more than 5 megadaltons. Of the 18 strains newly isolated from a vinegar factory, some of which were examined taxonomically, 17 contained plasmid DNA. The molecular weights of plasmids detected in this study were in the range of about one to over 17 megadaltons. The plasmids with molecular weights of less than 5 megadaltons were characterized with restriction endonucleases. The physical maps of two plasmids designated as pMV1O1 and pMV102, which were found in the isolated strains, were constructed.     

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.     

Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer.     

Dissemination of resistance plasmids among gentamicin-resistant enterobacteria from hospital patients.

Out of 184 patients who were infected or colonised with gentamicin-resistant enterobacteria, 17 (9%) harboured more than one strain. Single antibiotic-resistance plasmids were common to more than one of the different organisms isolated from nine patients, strongly suggestive of in-vivo conjugation. An "epidemic" plasmid with a molecular weight of approximately 110 megadaltons was found in 11 distinct strains isolated from four patients. Seven of the organisms harbouring this plasmid were Klebsiella aerogenes. Spread of multiple-resistance plasmids among endemic gentamicin-resistant enterobacteria is not uncommon, and these organisms provide a reservoir of plasmids that may ultimately spread to more pathogenic genera.     

Development of genetic techniques for the psychrotrophic fish pathogen Flavobacterium psychrophilum

Flavobacterium psychrophilum, a member of the Cytophaga-Flavobacterium-Bacteroides group, is an important pathogen of salmonid fish. Previous attempts to develop genetic techniques for this fastidious, psychrotrophic bacterium have met with failure. Here we describe the development of techniques for the genetic manipulation of F. psychrophilum and the identification of plasmids, selectable markers, a reporter system, and a transposon that function in several isolates of this fish pathogen. The antibiotic resistance genes ermF, cfxA, and tetQ function in F. psychrophilum. Cloning vectors based on the F. psychrophilum cryptic plasmid pCP1 which carried these selectable markers were introduced by conjugation from E. coli, resulting in antibiotic-resistant colonies of F. psychrophilum. Conjugative transfer of DNA into F. psychrophilum was strain dependent. Efficient transfer was observed for two of the seven strains tested (THC02-90 and THC04-90). E. coli lacZY functioned in F. psychrophilum when expressed from a pCP1 promoter, allowing its development as a reporter for studies of gene expression. Plasmids isolated from F. psychrophilum were efficiently introduced into F. psychrophilum by electroporation, but plasmids isolated from E. coli were not suitable for transfer by this route, suggesting the presence of a restriction barrier. DNA isolated from F. psychrophilum was resistant to digestion by Sau3AI and BamHI, indicating that a Sau3AI-like restriction modification system may constitute part of this barrier. Tn4351 was introduced into F. psychrophilum from E. coli and transposed with apparent randomness, resulting in erythromycin-resistant colonies. The techniques developed in this study allow for genetic manipulation and analysis of this important fish pathogen.     

A comparative analysis of the Salmonella typhi strains isolated from patients and bacterial carriers

The comparative analysis of 133 S. typhi clinical strains isolated from patients and carriers in Dnepropetrovsk Province in 1978-1987 was carried out. As shown by this analysis, 10 Vi phage types were represented in the set of strains under study, phage types A and F1 being the most numerous ones. Phage type F1 occurred less frequently among the strains isolated from carriers. 31.1% of the strains were found to contain plasmids with different molecular weight ranging from 96 to 0.5 MD. The occurrence of plasmid-containing strains remained at the same level during the whole period under study. Low-molecular plasmids occurred more frequently in the strains isolated from carriers. The minimal suppressive concentrations of a number of antibiotics, such as penicillin, ampicillin, monomycin, chloramphenicol, tetracycline, rifampicin and streptomycin, were determined. 7% of the strains were resistant to penicillin, 9% to monomycin, 15%--to tetracycline and 2.6% to chloramphenicol. The correlation between penicillin and monomycin resistance of the strains and the presence of the plasmid with a molecular weight of 60 MD in these strains was established. All strains were shown to be highly variable in the degree of their virulence: from 10(2) to 10(8). The strains isolated from patients possessed greater virulence.     

Polyresistant Enterobacteriaceae strains and their plasmids in a hospital. Medical and theoretical aspects

The properties and origin of multiple resistant strains of Enterobacteriaceae found in the intestine and nasopharynx of infants admitted to the hospital for premature infants were studied. The strains of E. coli of different serovars isolated at various periods contained similar conjugative R plasmids with a molecular weight of 80 Md belonging to the O incompatibility group controlling resistance to kanamycin and physically independent small plasmids controlling resistance to ampicillin (7 Md) and streptomycin-sulfanilamides (4 Md). Multiple drug resistance in the strains of K. pneumoniae was controlled by single large (100-120 Md) plasmid cointegrates with 6-8 resistance markers. Such cointegrates consisted of several potentially independent plasmids, sometimes dividing on transformation of plasmid DNA of the recipient strains of E. coli K12. The small plasmids controlling resistance to ampicillin and streptomycin-sulfanilamides similar to the respective plasmids of E. coli were the constant components of the plasmids cointegrates. The multiple drug resistance in the above strains was combined with high capacity for colonization in premature infants. The medical staff and mothers were the sources of bacterial strains with single plasmids controlling definite types of resistance. It is suggested that the multiple resistant strains of Enterobacteriaceae are formed in hospital as a result of accumulation of the plasmids or plasmid markers and selection. One of the conditions for successive acquisition of new plasmid markers by definite bacterial strains was their high capacity for colonization in patients, which provided constant contacts and genetic exchange of such strains with a wide range of immigrant strains during colonization in the newly admitted patients.