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PubMed1981

Plasmids coding for colonization factor antigen I and heat-stable enterotoxin production isolated from enterotoxigenic Escherichia coli: comparison of their properties.

We examined seven enterotoxigenic Escherichia coli strains which produced colonization factor antigen I (CFA/I). Four of these strains were from South Africa (three serotype O78:H12 and one serotype O63:H-), one was from Ethiopia (O78:H12), and two were from Bangladesh (O78:H11 and O78:H12). Plasmids coding for CFA/I were mobilized from six of these strains by using resistance or enterotoxin factors. No plasmid was mobilized from the serotype O78:H12 Bangladesh strain. The transconjugants obtained from crosses with the O78 strains also produced heat-stable enterotoxin (ST), and additional investigations showed that CFA/I and ST were coded for by a single non-autotransferring plasmid. These plasmids were fertility inhibition negative, did not restrict any of the coliphages with which they were tested, and were incompatible with each other. Four had molecular weights of approximately 60 X 10(6), and one had a molecular weight of 52 X 10(6). Like the other CFA/I plasmids, the CFA/I plasmid transferred from the O63:H- strain coded for ST, but this plasmid also coded for heat-labile enterotoxin. In most other respects the properties of this plasmid were similar to those of the CFA/I-ST plasmids previously described. The molecular weight of this plasmid was 65 X 10(6). The IncT R-factor Rtsl was marked with a transposon for tetracycline resistance and then transferred into the two Bangladesh wild-type strains. Plasmids which coded for tetracycline resistance, CFA/I, and ST were transferred from these strains. These plasmids were incompatible with Rtsl and with the CFA/I-ST plasmids described above and were recombinants between Rtsl and a CFA/I-ST plasmid. Their properties are also described.

Antigens, Bacterial↗
PubMed1987

Plasmid-associated virulence of Salmonella typhimurium.

We investigated the role of the 100-kilobase (kb) plasmid of Salmonella typhimurium in the virulence of this organism for mice. Three strains, LT2-Z, SR-11, and SL1344, which possessed 100-kb plasmids with identical restriction enzyme digestion profiles, were cured of their respective 100-kb plasmids after Tnmini-tet was used to label plasmids. Curing wild-type virulent strains SR-11 and SL1344 raised peroral 50% lethal doses from 3 x 10(5) and 6 x 10(4) CFU, respectively, to greater than 10(8) CFU. Both wild-type strains had intraperitoneal 50% lethal doses of less than 50 CFU, whereas the intraperitoneal 50% lethal doses for cured SR-11 and SL1344 were less than 50 and 400 CFU, respectively. Reintroduction of the Tnmini-tet-labeled, 100-kb plasmid restored wild-type virulence. Invasion from Peyer's patches to mesenteric lymph nodes and spleens after peroral inoculation was the stage of pathogenesis most affected by curing S. typhimurium of the 100-kb plasmid. Wild-type S. typhimurium replicated in spleens of mice inoculated intravenously to a greater extent than did plasmid-cured derivatives. Wild-type and cured strains equally adhered to and invaded Henle-407, HEp-2, and CHO cells; furthermore, the presence of the 100-kb plasmid was not necessary for replication of S. typhimurium within CHO cells. The 100-kb plasmid had no effect on phagocytosis and killing of S. typhimurium by murine peritoneal macrophages in vitro and in vivo. Similarly, wild-type and plasmid-cured strains were resistant to killing by 90% normal human, rabbit, and guinea pig sera. All wild-type and plasmid-cured S. typhimurium strains possessed complete lipopolysaccharide, as determined by silver staining solubilized cells in sodium dodecyl sulfate-polyacrylamide gels. We have confirmed the role of the 100-kb plasmid of S. typhimurium in virulence, primarily in invasion to mesenteric lymph nodes and spleens after peroral inoculation of mice. Involvement of the 100-kb plasmid in infection of mesenteric lymph nodes and spleens suggests a role for the plasmid in the complex interaction of S. typhimurium with cells of the reticuloendothelial system.

Animals↗
PubMed1987

Identification of self-transmissible plasmids in four Bacillus thuringiensis subspecies.

The transfer of plasmids by mating from four Bacillus thuringiensis subspecies to Bacillus anthracis and Bacillus cereus recipients was monitored by selecting transcipients which acquired plasmid pBC16 (Tcr). Transcipients also inherited a specific large plasmid from each B. thuringiensis donor at a high frequency along with a random array of smaller plasmids. The large plasmids (ca. 50 to 120 megadaltons), pXO13, pXO14, pXO15, and pXO16, originating from B. thuringiensis subsp. morrisoni, B. thuringiensis subsp. toumanoffi, B. thuringiensis subsp. alesti, and B. thuringiensis subsp. israelensis, respectively, were demonstrated to be responsible for plasmid mobilization. Transcipients containing any of the above plasmids had donor capability, while B. thuringiensis strains cured of each of them were not fertile, indicating that the plasmids confer conjugation functions. Confirmation that pXO13, pXO14, and pXO16 were self-transmissible was obtained by the isolation of fertile B. anthracis and B. cereus transcipients that contained only pBC16 and one of these plasmids. pXO14 was efficient in mobilizing the toxin and capsule plasmids, pXO1 and pXO2, respectively, from B. anthracis transcipients to plasmid-cured B. anthracis or B. cereus recipients. DNA-DNA hybridization experiments suggested that DNA homology exists among pXO13, pXO14, and the B. thuringiensis subsp. thuringiensis conjugative plasmids pXO11 and pXO12. Matings performed between strains which each contained the same conjugative plasmid demonstrated reduced efficiency of pBC16 transfer. However, in many instances when donor and recipient strains contained different conjugative plasmids, the efficiency of pBC16 transfer appeared to be enhanced.

Bacillus anthracis↗
PubMed2006

Complete sequencing and diversity analysis of the enterotoxin-encoding plasmids in Clostridium perfringens type A non-food-borne human gastrointestinal disease isolates.

Enterotoxin-producing Clostridium perfringens type A isolates are an important cause of food poisoning and non-food-borne human gastrointestinal diseases, e.g., sporadic diarrhea (SPOR) and antibiotic-associated diarrhea (AAD). The enterotoxin gene (cpe) is usually chromosomal in food poisoning isolates but plasmid-borne in AAD/SPOR isolates. Previous studies determined that type A SPOR isolate F5603 has a plasmid (pCPF5603) carrying cpe, IS1151, and the beta2 toxin gene (cpb2), while type A SPOR isolate F4969 has a plasmid (pCPF4969) lacking cpb2 and IS1151 but carrying cpe and IS1470-like sequences. By completely sequencing these two cpe plasmids, the current study identified pCPF5603 as a 75.3-kb plasmid carrying 73 open reading frames (ORFs) and pCPF4969 as a 70.5-kb plasmid carrying 62 ORFs. These plasmids share an approximately 35-kb conserved region that potentially encodes virulence factors and carries ORFs found on the conjugative transposon Tn916. The 34.5-kb pCPF4969 variable region contains ORFs that putatively encode two bacteriocins and a two-component regulator similar to VirR/VirS, while the approximately 43.6-kb pCPF5603 variable region contains a functional cpb2 gene and several metabolic genes. Diversity studies indicated that other type A plasmid cpe+/IS1151 SPOR/AAD isolates carry a pCPF5603-like plasmid, while other type A plasmid cpe+/IS1470-like SPOR/AAD isolates carry a pCPF4969-like plasmid. Tn916-related ORFs similar to those in pCPF4969 (known to transfer conjugatively) were detected in the cpe plasmids of other type A SPOR/AAD isolates, as well as in representative C. perfringens type B to D isolates carrying other virulence plasmids, possibly suggesting that most or all C. perfringens virulence plasmids transfer conjugatively.

Clostridium Infections↗
PubMed1989

Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication.

The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells. The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence. The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites. Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element. The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells. The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements. EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays. Plasmids with just two or six copies of these repeated elements failed to generate drug-resistant colonies at a normal level, and normal episomal plasmids were not detected in the resulting colonies. Rare colonies of cells resulting from transfection of these two- or six-copy mutants contained rearranged, episomal forms of the input plasmids. The rearrangements most often produced head-to-tail oligomers containing a minimum of eight 30-bp repeated elements. The rearranged plasmids were shown to be revertant for plasmid maintenance in that they yielded wild-type or greater numbers of drug-resistant colonies and persisted at the wild-type or a greater episomal copy number. By use of an EBV plasmid that contained no 30-bp elements, no revertants could be isolated. One to five copies of a synthetic linker corresponding to a consensus 30-bp repeated element inserted into a plasmid with no 30-bp elements now permitted the generation of oligomeric, episomal forms of the mutant test plasmid. These experiments demonstrate a requirement for a minimal number (six to eight copies) of the 30-bp repeated element. Deletions in the 65-bp dyad region had little or no effect upon the ability to generate enhanced numbers of drug-resistant D98-Raji colonies, indicating that the 30-bp repeated element is predominantly required for this phenotype.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed2005

Effect of naked eukaryotic expression plasmid encoding rat augmenter of liver regeneration on acute hepatic injury and hepatic failure in rats.

AIM: To study the protective effect of eukaryotic expression plasmid encoding augmenter of liver regeneration (ALR) on acute hepatic injury and hepatic failure in rats. METHODS: The PCR-amplified ALR gene was recombined with pcDNA3 plasmid, and used to treat rats with acute hepatic injury. The rats with acute hepatic injury induced by intraperitoneal injection of 2 mL/kg 50% carbon tetrachloride (CCl(4)) were randomly divided into saline control group and recombinant pcDNA3-ALR plasmid treatment groups. Recombinant pcDNA3-ALR plasmid DNA (50 or 200 microg/kg) was injected into the rats with acute hepatic injury intravenously, intraperitoneally, or intravenously and intraperitoneally in combination 4 h after CCl(4) administration, respectively. The recombinant plasmid was injected once per 12 h into all treatment groups four times, and the rats were decapitated 12 h after the last injection. Hepatic histopathological alterations were observed after HE staining, the expression of proliferating cell nuclear antigen (PCNA) in liver tissue was detected by immunohistochemical staining, and the level of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) was determined by biochemical method. The recombinant plasmid DNA (200 microg/kg) and saline were intraperitoneally injected into the rats with acute hepatic failure induced by intraperitoneal injection of 4 mL/kg 50% CCl(4) after 4 h of CCl(4) administration, respectively. Rats living over 96 h were considered as survivals. RESULTS: The sequence of ALR cDNA of recombinant pcDNA3-ALR plasmid was accordant with the reported sequence of rat ALR cDNA. After the rats with acute hepatic injury were treated with recombinant pcDNA3-ALR plasmid, the degree of liver histopathological injury markedly decreased. The pathologic liver tissues, in which hepatic degeneration and necrosis of a small amount of hepatocytes and a large amount of infiltrating inflammatory cells were observed, and they became basically normal in the most effective group after four times of injection of recombinant pcDNA3-ALR plasmid. The indexes of PCNA significantly increased in the recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The level of serum AST and ALT remarkably reduced in recombinant pcDNA3-ALR plasmid treatment groups compared to model group. The results showed that the effect of 200 microg/kg recombinant pcDNA3-ALR plasmid in the rats with acute liver injury was stronger than that of 50 microg/kg pcDNA3-ALR DNA. The effect of intravenous injection of recombinant pcDNA3-ALR plasmid was better. After the rats with acute hepatic failure were treated with recombinant pcDNA3-ALR plasmid, the survival rate (40%) significantly increased in treatment groups compared to control group (15%, P<0.01). CONCLUSION: The ALR gene may play an important role in relieving acute hepatic injury and hepatic failure by promoting hepatic cell proliferation and reducing level of AST and ALT in CCl(4)-intoxicated rats.

Animals↗
PubMedDate not supplied

[Analysis of the plasmid profile of various Salmonella serotypes].

INTRODUCTION: Every year foodborne infections cause millions of illnesses but many of them go undiagnosed and unreported. The epidemiology of these illnesses is changing, new pathogens have emerged (Escherichia coli O157:H7, Cyclospora cayetanensis, Vibrio vulnificus). Salmonella spp. is the most common bacterial cause of acute enterocolitis with us. All over the world, as well as in our country the most often isolated serotype is Salmonella Enteritidis. A great problem in many countries is the multiresistant Salmonella Typhimurium, as well as other serotypes resistant to a great number of antimicrobial drugs (S. Hadar, S. Typhi). Clinical microbiologists are often asked to determine the relatedness of bacterial isolates. Recently, traditional methods of strain typing such as bacteriophage typing, resistotyping and serotyping, have been supplemented or replaced in many laboratories with newer molecular methods such as plasmid fingerprinting, ribotyping. PCR-based methods, etc. The goal of strain typing is to provide evidence that epidemiologically related isolates collected during an outbreak are also genetically related and thus represent the same strain. MATERIAL AND METHODS: In the laboratory for Enterobacteriaceae of the Institute of Public Health Novi Sad in the four year period (1995-1998) 3659 primary isolates of Salmonella spp. were isolated using standard bacteriological methods (cultural, biochemical and serological examination). For certain strain of Salmonella Enteritidis, S. Typhimurium and S. Hadar susceptibility to antimicrobial agents was tested by disc-diffusion test (Kirby-Bauer) and plasmid profiles were analyzed. Plasmid DNA was extracted by Birnboim and Doly alkaline lysis method and plasmid bands were separated by electrophoresis in agarose gel. RESULTS: In the period of 1995-1998 the most common serotype isolated was Salmonella Enteritidis with 3017 (82.5%) of the total number of isolated Salmonellas; S. Typhimurium 203 (5.5%), S. Hadar 118 (3.2%). Plasmid profiles were tested in 10 S. Enteritidis isolates that originated from patients with sporadic cases of diarrhea. All investigated strains had one plasmid band with molecular weight of 38 MDa. All isolates were susceptible to all antibiotics tested. Ten isolates of S. Hadar originated from one outbreak from food samples and stools of patients with diarrheal disease and from the worker in the restaurant. All isolates were resistant to ampicillin and streptomycin, and plasmid profile analysis showed 5 plasmid bands with molecular weights of 13, 5.4, 4.2, 2.0 and 1.7 MDa. Chosen strains of S. Typhimurim were not epidemically related. Strains number 1, 3, 4, 5 were susceptible to all antibiotics tested, and had only one plasmid of 50 MDa, strain number 7 was resistant to streptomycin and had 2 plasmid bands of 50 and 1.7 MDa, other strains were multiresistant and had different plasmid profiles with 4-7 plasmid bands with molecular weights ranging from 50-1.4 MDa. CONCLUSION: Plasmid profile analysis is not a sufficient method for examination of Salmonella Enteritidis which is the most common cause of enterocolitis with us. It is, however, a helpful method for proving epidemiological and clonal relatedness of Salmonella isolates that are resistant to antimicrobial agents and have a great number of plasmids (such as some strains of S. Typhimurim and S. Hadar).

Enterocolitis↗
PubMed1981

Interspecific plasmid and genomic DNA sequence homologies and localization of nif genes in effective and ineffective strains of Rhizobium japonicum.

Three strains of Rhizobium japonicum were examined for the presence of interspecific conserved plasmid-borne DNA sequences and the location of their nif DNA sequences. Strains 61A76 and 110, which both form effective (nitrogen fixing) nodules on soybeans show very low (24%) total DNA sequence homology with each other; strain 61A76 contains one plasmid, and strain 110 contains no identifiable plasmids. Strain 61A24 which forms ineffective nodules on soybeans shows relatively high (50%) sequence homology to strain 110 and contains two plasmids. While the total sequence homology between purified plasmid DNA isolated from strains 61A76 and 61A24 appears limited, heterologous hybridizations between plasmids and total DNAs from these strains and DNA from strain 110 suggest that several sequences which are plasmid-borne in one strain can occur in the chromosome or in very large plasmid(s) in the other strains. The plasmid isolated from strain 61A24 exhibits some homology with several SmaI fragments of an octopine Ti plasmid from Agrobacterium tumifaciens, whereas the 61A76 plasmid shows no homology to this Ti plasmid. DNA sequences from five strains of Rhizobium japonicum were found to hybridize with pSA30, a plasmid carrying the Klebsiella pneumoniae nif-KDH genes, but these sequences were not located on the isolated plasmids. The organization of these genes appears to be similar in strains 110 and 61A24, but is different in strain 61A76.

Base Sequence↗
PubMed1997

Viability during storage and stability of plasmids during storage and subculturing in strains of Vibrio anguillarum.

The stability of plasmids, 3.3 kb - approximately 200 kb, in 8 strains of Vibrio anguillarum displaying different plasmid profiles and reactions with O-antisera was investigated over an 18 months period. All plasmid profiles proved to be resistant to storage at different temperatures but strains stored at 37 degrees C were only viable for a short period of maximum 2 months. Strains stored at 5 degrees C and 20 degrees C were viable for a longer period. Viable strains maintained their plasmid profile throughout the experiment, except in 2 cases where a 67 kb and a 200 kb plasmid were lost. Strains stored at -80 degrees C all remained viable and maintained their plasmid profile throughout the study. By subcultivating daily for up to 100 successive days, most strains maintained their plasmid profiles. Only 2 strains lost their plasmid. When picking 100 single colonies from agar plates, none of the colonies showed plasmid profiles deviating from the expected. The results suggest that plasmid profiles among V. anguillarium are very stable during subculturing, storage and laboratory handling using standard laboratory procedures, and thus, reliable for epidemiological investigations. In a second experiment, 2 pairs of 2 strains were grown together in mixed cultures. They were identical in all traits, except that one strain in each pair harboured the 67 kb pJM1-like virulence plasmid, whereas the other had lost this plasmid. The result showed that the growth rate was the same for strains with and without the plasmid, indicating that under laboratory conditions, this plasmid is neither a benefit nor the opposite for bacterial growth.

Animals↗
PubMedDate not supplied

Plasmid profile analysis and antibiotic resistance of Salmonella strains from clinical isolates in Cluj-Napoca.

Resistance patterns, plasmid profiles and the genetic resistance determinants were investigated in 38 isolates of Salmonella enterica serotype Typhimurium and 19 isolates of Salmonella enterica serovar Enteritidis derived from children hospitalized in two clinics in Cluj-Napoca, during the period of 1995-1997. Incidence of plasmid and antibiotic resistance was very high in Salmonella typhimurium isolates. All strains were resistant to almost all antibiotics tested but susceptible to the third generation cephalosporines and fluoroquinolones. We identified three resistance patterns and six plasmid profiles. Each plasmid profile was characterized by the presence of two large plasmids of 150-180 Kbp. Approximately 60% of strains harbored three or four small plasmids of 1.3 to 9.5 Kbp. The plasmids of 8.5 Kbp encoded resistance to beta-lactam antibiotics and were non-conjugative. The other small plasmids were cryptic and also non-conjugative. Salmonella enteritidis isolates were susceptible to many antibiotics, except Tetracycline and Trimethoprim-Sulfamethoxazole. We identified three different resistance patterns but nine plasmid profiles. All plasmid profiles were characterized by the presence of a large plasmid (> 100 Kbp). The number and the diversity of small plasmids were higher than in S. typhimurium strains. There was no parallelism between resistance and plasmid profile: for the same resistance pattern a number of two or three plasmid profiles were found. Our conclusions are that Salmonella typhimurium strains were multiresistant to antibiotics and that many genetically different strains of Salmonella typhimurium and Salmonella enteritidis were responsible for gastroenteritis in children from Cluj County. The increasing antibiotic resistance highlights the need for more refined methods in genetic and epidemiological characterization of bacteria involved in gastrointestinal infections.

Anti-Bacterial Agents↗
PubMedDate not supplied

[Shigellae isolated in 1997--plasmid profiles and antibiotic resistance].

INTRODUCTION: Shigella spp is one of the most frequently isolated bacteria causing acute diarrhea with us. Genetics of pathogenicity of Shigella spp. includes chromosomal and plasmid genes. Most virulence factors are coded by invasion plasmid antigen genes residing on a 180-230 MDa plasmid. There is a big problem with multiple resistance of Shigella spp. strains, which is mostly plasmid-borne. Genetic analysis of bacterial cells, that is plasmid profile analysis, is important for investigation of sources and ways of spreading of the infection. All isolates originating from the same clone have identical plasmid profiles, i.e. number and size of plasmids. The aim of the investigation was: comparing the type of resistance to antimicrobical agents found in epidemic and nonepidemic. Shigella strains isolated in 1997, analyzing plasmid profiles of these isolates and confirming their epidemic connection. MATERIAL AND METHODS: Susceptibility to antibiotics was examined by a standard disc-diffusion method. Plasmid profiles of 40 strains (20 from the outbreak and 20 from sporadic cases) were tested using a method of alkaline lysis by Birnboim and Doly followed by electrophoresis in agarose gel. RESULTS: Shigella strains were resistant to antimicrobial agents which are most commonly used. Epidemic isolates shared the same resistance type, they were resistant to cephalexin, streptomycin and co-trimoxazole. The dominant type of resistance of nonepidemic strains was to ampicillin, streptomycin and co-trimoxazole. Strains isolated during the outbreak had identical plasmid profiles (2 plasmid bands of 55 and 1.5 MDa). Non-epidemic isolates had different plasmid profiles as well as type of resistance. CONCLUSION: Strains of Shigella spp. isolated during an outbreak had the same type of resistance and the same plasmid profiles, which indicated their origin from the same clone. The plasmid profile analysis is a reliable and precise method for determination of epidemic connection of Shigella isolates.

DNA, Bacterial↗
PubMed1998

Activity and stability of a recombinant plasmid-borne TCE degradative pathway in biofilm cultures

The activity and stability of the TCE degradative plasmid TOM31c in the transconjugant host Burkholderia cepacia 17616 was studied in selective and non-selective biofilm cultures. The activity of plasmid TOM31c in biofilm cultures was measured by both TCE degradative studies and the expression of the Tom pathway. Plasmid loss was measured using continuous flow, rotating annular biofilm reactors, and various analytical and microbiological techniques. The probability of plasmid loss in the biofilm cultures was determined using a non-steady-state biofilm plasmid loss model that was derived from a simple mass balance, incorporating results from biofilm growth and plasmid loss studies. The plasmid loss model also utilized Andrew's inhibition growth kinetics and a biofilm detachment term. Results from these biofilm studies were compared to similar studies performed on suspended cultures of Burkholderia cepacia 17616-TOM31c to determine if biofilm growth has a significant effect on either plasmid retention or Tom pathway expression (i.e., TCE degradation rates). Results show that the activity and expression of the Tom pathway measured in biofilm cultures was significantly less than that found in suspended cultures at comparable growth rates. The data obtained from these studies fit the plasmid loss model well, providing plasmid loss probability factors for biofilm cultures that were equivalent to those previously found for suspended cultures. The probability of plasmid loss in the B. cepacia 17616-TOM31c biofilm cultures was equivalent to those found in the suspended cultures. The results indicate that biofilm growth neither helps nor hinders plasmid stability. In both the suspended and the biofilm cultures, plasmid retention and expression could be maintained using selective growth substrates and/or an appropriate plasmid-selective antibiotic. Copyright 1998 John Wiley & Sons, Inc.

Journal Article↗
PubMed1993

Plasmid diversity in Escherichia coli isolated from processed poultry and poultry processors.

Plasmids of bacteria selected from different bacterial populations because they shared a distinctive antimicrobial resistance phenotype have sometimes had identical restriction fragments. Such identical plasmids are thought to belong to small and thus epidemic clones because the plasmid content of unselected resistant isolates has seemed diverse. To survey this presumed diversity and its implications for the lineage of resistance plasmids we examined the transferability, sizes and EcoR1 restriction fragment sizes of plasmids in both Escherichia coli isolated randomly from poultry raised by 16 growers as they were being processed through two plants and in isolates from the urine of women processing poultry in those plants. Forty two (24%) of 175 resistant isolates from poultry of 16 growers and 9 (26%) of 34 resistant isolates from the poultry processors transferred resistance conjugatively to varied combinations of antimicrobials. No poultry isolate had both the same expressed and the same transferred combination as any processor's isolate. The DNA bands which could be discerned in electrophoresis gels of restricted or unrestricted plasmid extracts of isolates or their transconjugants from 156 of the poultry and 24 of the poultry processors appeared diverse. Pairs of related-appearing plasmids were seen in consecutive isolates of poultry from each of two growers and in one pair from different growers. One set of identical-appearing plasmids was seen in 3 consecutive isolates from poultry of one grower, others in 2 consecutive isolates from a second grower's poultry, in 2 non-consecutive isolates of a third grower's, and in single isolates from poultry of 2 different growers. None of the plasmids from any of the human isolates appeared related to those from any other human isolate or to those of any poultry isolate. These results indicate that resistance plasmids are highly diverse and that all but two of the exceptions to complete diversity in the isolates surveyed here could be ascribed to cross colonization within flocks of individual poultry growers. Also, while none of the plasmids in the poultry isolates appeared ancestral to any of plasmids in the poultry processors' isolates, their diversity indicates that those sampled plasmids would be only a very small fraction of the total number of different plasmids in bacteria colonizing poultry processed at that time or earlier.

Animals↗
PubMed1980

Genetical relationship between R plasmids derived from Salmonella and Escherichia coli obtained from a pig farm, and its epidemiological significance.

A total of 475 Salmonella strains belonging to 5 serovars, isolated from a pig farm which had been heavily contaminated with Salmonella for the past 2 years were tested for antibiotic susceptibility and detection of R plasmids. Thirty-three Escherichia coli isolates from the same farm were also examined in a similar way. Out of 475 strains 348 (73.2%) were resistant to one or more antibiotics such as tetracycline (Tc), streptomycin (Sm), sulfadimethoxine (Su), chloramphenicol (Cm) and kanamycin (Km), and 247 (85.2%) out of 290 strains belonging to 3 serovars examined harboured conjugative R plasmids. There was no change in the pattern of drug resistance during this survey nor any variation in the pattern of resistance of R plasmids, whatever the serovar. The antibiogram pattern Tc Sm Su, mainly S. typhimurium, was common among Salmonella strains. Among the transferred resistance patterns, the thermosensitive R plasmids conferring the Tc marker detected in this study were Fi-, and belonged to incompatibility group H1, whereas the R plasmids conferring Sm Su resistances which coexisted in the same host were Fi+, and compatible with the reference R plasmids tested. The I alpha plasmid conferring Cm resistance alone was isolated from S. anatum and the FII plasmid conferring Sm Km resistances was also isolated from S. typhimurium. In contrast, the 33 E. coli strains examined were resistant to three or five antibiotics and most of the resistance markers were located on conjugative R plasmids. I alpha plasmids conferring Cm resistance alone or FII plasmids conferring Cm or Km markers were common in the E. coli strains. H1 and H2 plasmids conferring multiple resistance markers were also found in them. The genetic properties of R plasmids derived from Salmonella or E. coli strains are compared, and the potential spread of R plasmids between strains of Salmonella and E. coli is discussed.

Animals↗
PubMed1995

Comparison of ccd of F, parDE of RP4, and parD of R1 using a novel conditional replication control system of plasmid R1.

A number of plasmid-encoded gene systems are thought to stabilize plasmids by killing plasmid-free cells (also termed post-segregational killing or plasmid addiction). Here we analyse the mechanisms of plasmid stabilization by ccd of F, parDE of RP4 and parD of R1, and compare them to hok/sok of R1. To induce synchronous plasmid loss we constructed a novel plasmid replication-arrest system, which possesses the advantage that plasmid replication can be completely arrested by the addition of IPTG, a non-metabolizable inducer. Using isogenic plasmid constructions we have found, for the first time, consistent correlation between the effect on steady-state loss rates and the effect on cell proliferation in the plasmid replication-arrest assay for all three systems. The parDE system had the most pronounced effect both on plasmid stabilization and on plasmid retention after replication arrest. In contrast, ccd and parD both exhibited weaker effects than anticipated from previously published results. Thus, our results indicate that the function and efficiencies of some of the systems should be reconsidered. Our results are consistent with the previously postulated hypothesis that ccd and parDE act by killing plasmid-free segregants, whereas parD seems to act by inhibiting cell division of plasmid-free segregants.

Bacterial Proteins↗
PubMed1976

In vivo transcription of R-plasmid deoxyribonucleic acid in Escherichia coli strains with altered antibiotic resistance levels and/or conjugal proficiency.

The amounts of plasmid deoxyribonucleic acid (DNA) and the levels of the in vivo transcription of the Escherichia coli plasmids R538-1 (repressed for conjugal transfer) and R538-1drd (derepressed for transfer) were determined by DNA-DNA hybridization and DNA-ribonucleic acid hybridization, respectively. The results demonstrate that the level of plasmid transcription is increased by two-fold in the strain carrying the derepressed plasmid, compared to an isogenic strain carrying the repressed plasmid, whereas the amount of plasmid DNA is approximately the same, suggesting that the transfer genes are under transcriptional control. Levels of plasmid DNA, plasmid DNA transcription, and chloramphenicol acetyltransferase activity were also compared in a mutant strain that carried the R538-1drd plasmid and was resistant to high levels of antibiotics. This strain produces about 13 copies of plasmid DNA per chromosome compared to five copies for the parent strain. The level of transcription of plasmid DNA was found to be twofold higher in the high-level resistant strain, whereas the level of chloramphenition, acetyltransferase activity was increased by 10-fold. In addition the levels of plasmid DNA transcription and chloramphenicol acetyltransferase activity in the high-level resistant strain were found to be further increased by the presence of high levels of chloramphenicol in the growth medium. The amount of plasmid DNA remained constant under these conditions, indicating that high levels of chloramphenicol can stimulate the expression of plasmid genes at the level of transcription in this strain.

Acetyltransferases↗
PubMed1980

Control of plasmid R1 replication: functions involved in replication, copy number control, incompatibility, and switch-off of replication.

A small derivative of plasmid R1 was used to integratively suppress a chromosomal dnaA(Ts) mutation. The strain obtained grew normally at 42 degrees C. The integratively suppressed strain was used as recipient for various plasmid R1 derivatives. Plasmid R1 and miniplasmid derivatives of R1 could be established in the strain that carried an integrated R1 replicon, but they were rapidly lost during growth. However, plasmids also carrying ColE1 replication functions were almost completely stably inherited. The integratively suppressed strain therefore allows the establishment of bacteria diploid with respect to plasmid R1 and forms a useful and sensitive system for studies of interaction between plasmid R1 replication functions. Several of the chimeric plasmids caused inhibition of growth at high temperatures. All plasmids that inhibited growth carried one particular PstI fragment from plasmid R1 (the PstI F fragment), and in all cases the growth inhibition could be ascribed to repression of initiation of chromosome replication at 42 degrees C, i.e., they carry a trans-acting switch-off function. Furthermore, the analogous PstI fragments from different copy mutants of plasmid R1 were analyzed similarly, and one mutant was found to lack the switch-off function. The different chimeric plasmids were also tested for their incompatibility properties. All plasmids that carried the switch-off function (and no other plasmids) also carried R1 incompatibility gene(s). Since the PstI F fragment, which is present on all these plasmids, is very small (0.35 x 10(6)), it is suggested that the switch-off regulation of replication (by an inhibitor), incompatibility, and copy number control are governed by the same gene.

Chromosomes, Bacterial↗
PubMed1988

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.

Conjugation, Genetic↗