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Results for “plasmid analysis”

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PubMed1994

Arbitrarily primed PCR, ribotyping, and plasmid pattern analysis applied to investigation of a nosocomial outbreak due to Enterobacter cloacae in a neonatal intensive care unit.

In December 1992, Enterobacter cloacae was isolated from the oropharynx and respiratory tract of six ventilated neonates hospitalized in the intensive care unit (ICU) of our hospital. To establish the spread of the outbreak, 41 strains of E. cloacae were analyzed for genotypic markers by three methods: plasmid profile analysis, ribotyping with EcoRI or PvuII endonuclease, and arbitrarily primed (AP) PCR. The tested strains included 12 isolates from the 6 epidemic cases, 4 isolates from the respiratory tract of 4 children hospitalized in other wards during the same period, 13 isolates from 12 children hospitalized in pediatric units before or after the outbreak, and 12 epidemiologically unrelated isolates. Ribotyping and AP PCR demonstrated that each of the last 12 strains exhibited distinct genomic patterns, as did each of the strains isolated from neonates hospitalized before or after the epidemic peak. Conversely, two clones of strains were found among the isolates recovered in December, with concordant results being obtained by the three typing methods: the first clone included seven strains from five ventilated children in the ICU and two children from another ward; another clone was shared by one neonate in the ICU and an infant from another ward. These results indicate that ribotyping and AP PCR-the latter applied, to our knowledge, for the first time to the genotypic analysis of E. cloacae--represent very discriminatory tools for the investigation of nosocomial outbreaks caused by this species.

Bacterial Typing Techniques↗

PubMed1988

Molecular analysis of plasmid DNA repair within ultraviolet-irradiated Escherichia coli. II. UvrABC-initiated excision repair and photolyase-catalyzed dimer monomerization.

In this study, a novel approach to the analysis of DNA repair in Escherichia coli was employed which allowed the first direct determination of the mechanisms by which endogenous DNA repair enzymes encounter target sites in vivo. An in vivo plasmid DNA repair analysis was employed to discriminate between two possible mechanisms of target site location: a processive DNA scanning mechanism or a distributive random diffusion mechanism. The results demonstrate that photolyase acts by a distributive mechanism within E. coli. In contrast, UvrABC-initiated excision repair occurs by a limited processive DNA scanning mechanism. A majority of the dimer sites on a given plasmid molecule were repaired prior to the dissociation of the UvrABC complex. Furthermore, plasmid DNA repair catalyzed by the UvrABC complex occurs without a detectable accumulation of nicked plasmid intermediates despite the fact that the UvrABC complex generates dual incisions in the DNA at the site of a pyrimidine dimer. Therefore, the binding or assembly of the UvrABC complex on DNA at the site of a pyrimidine dimer represents the rate-limiting step in the overall process of UvrABC-initiated excision repair in vivo.

DNA Repair↗

PubMedDate not supplied

[Restriction analysis of plasmid of the strain Streptomyces sp.27].

Plasmid pSS27 was found in streptomycete strain isolated from the soil sample in the Kyiv region. The paper deals with the data of restriction analysis of streptomycete plasmid pSS27 and its molecular weight. Plasmid pSS27 has unique restriction sites for endonucleases BglII and EcoRI, there are also the restriction sites for SalGI (3 sites), PstI (4 sites), BglI (5 sites), Bsp120I (7 sites) and SacII (11 sites). The molecular weight of plasmid DNA pSS27, being equal to 13.7 +/- 0.2 kb has been determined as a result of its restriction analysis.

Animals↗

PubMed1994

A nosocomial outbreak due to non-encapsulated Haemophilus influenzae: analysis of plasmids coding for antibiotic resistance.

An outbreak of infections with non-encapsulated Haemophilus influenzae, resistant to ampicillin, chloramphenicol, sulphonamide and tetracycline involved 13 elderly patients and three nurses on acute admission and care of the elderly wards. Thirty-two isolates were found to be indistinguishable on analysis of biotype, antibiogram, serotype and major outer membrane proteins (MOMP). Plasmids could not be identified in the original isolates but after mating with a Rec A H. influenzae recipient, the resultant transconjugates were found to harbour either a 72 kilobase pair (kB) plasmid coding for resistance to chloramphenicol, ampicillin, sulphonamide and tetracycline or a 65 kB plasmid coding for resistance to chloramphenicol, ampicillin and sulphonamide. Both plasmids yielded virtually indistinguishable restriction digest patterns. This suggests that the tetracycline resistance gene (Tc gene) is a non-essential component of one basic plasmid responsible for the multiple antibiotic resistances seen in the strains recovered during the outbreak. This illustrates the value of plasmid profiles to compare strains of non-encapsulated H. influenzae, and suggests that plasmid restriction enzyme analysis is critical.

Aged↗

PubMed1996

Molecular analysis of closely related copper- and streptomycin-resistance plasmids in Pseudomonas syringae pv. syringae.

The genetic relationship of a group of copper (Cur) and streptomycin (Smr) resistance plasmids and their Pseudomonas syringae pv. syringae hosts was examined. Each of these plasmids contained sequences homologous to the oriV and par sequences from pOSU900, a cryptic P. syringae pv. syringae plasmid. Analysis of restriction digest patterns of plasmid DNA indicated that the plasmids could be clustered into four groups; two of the groups contained multiple members which differed by only a few fragments. An analysis of the host P. syringae genotypes using the arbitrarily primed PCR technique and genomic DNA indicated that the host strains could be placed in groups similar to those resulting from analysis of plasmid DNA. Southern hybridization analyses of plasmid DNA indicated that each Smr plasmid contained sequences homologous to probes specific for the strA-strB Smr genes and the transposase and resolvase genes from Tn5393. All plasmids hybridized to two additional probes derived from P. syringae plasmid DNA, but none of the plasmids contained IS51 or IS801 sequences. Furthermore, Tn5393 was mobilized, presumably by transposition, between the incompatible plasmids pPSR5 and pPSR4 in P. syringae pv. syringae FF5. The variation in molecular structure of the closely related plasmids in this study is similar to that observed with antibiotic-resistance plasmids from clinical bacteria.

Copper↗

PubMed2000

Analysis of plasmid and chromosomal DNA of multidrug-resistant Salmonella enterica serovar typhi from Asia.

Molecular analysis of chromosomal DNA from 193 multidrug-resistant (MDR) Salmonella enterica serovar Typhi isolates from 1990 to 1995 from Pakistan, Kuwait, Malaysia, Bangladesh, and India produced a total of five major different pulsed-field gel electrophoresis (PFGE) patterns. Even within a particular country MDR S. enterica serovar Typhi DNA was found to be in different PFGE groups. Similar self-transferable 98-MDa plasmids belonging to either incompatibility group incHI1 or incHI1/FIIA were implicated in the MDR phenotype in S. enterica serovar Typhi isolates from all the locations except Quetta, Pakistan, where the majority were of incFIA. A total of five different PFGE genotypes with six different plasmids, based on incompatibility and restriction endonuclease analysis groups, were found among these MDR S. enterica serovar Typhi isolates.

Anti-Bacterial Agents↗

PubMed1995

Restriction endonuclease analysis of plasmid DNA of Yersinia pseudotuberculosis infections in Shimane Prefecture, Japan.

The epidemiology of Yersinia pseudotuberculosis infections in a limited area of Shimane Prefecture, Japan, was examined by serotyping and restriction endonuclease analysis of virulence plasmid DNA of Y. pseudotuberculosis strains isolated from humans, wildlife animals and river water. Almost all isolates from three sources belonged to serotype 1b REAP pattern D and serotype 4b REAP patterns B, G and L. The identity of the distribution of serotype and REAP patterns among isolates from humans, wildlife animals and river water shows that Y. pseudotuberculosis is transmitted to humans through environmental substances contaminated by wildlife animals infected with this species.

Animals↗

PubMed2001

Comparative sequence analysis of plasmids pME2001 and pME2200 of methanothermobacter marburgensis strains Marburg and ZH3.

Comparison of the updated complete nucleotide sequences of the two related plasmids pME2001 and pME2200 from the thermophilic archaeon Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum) strains Marburg and ZH3, respectively, revealed an almost identical common backbone structure and five plasmid-specific inserted fragments (IFs), four of which are flanked by perfect or nearly perfect direct repeats 25-52 bp in length. A 4354-bp minimal replicon was derived from the alignment of the two plasmids, which encodes one putative antisense RNA related to replication control and five open reading frames (ORFs) organized in two operons. The first operon consists of four ORFs, the third of which, i.e. ORF3, contains a helix-turn-helix motif and a purine NTP-binding motif often found in proteins involved in DNA metabolic processes. The database search results suggest that ORF3 might function as a replication initiator protein. The large putative Rep protein encoded by pME2001 was overexpressed in Escherichia coli as an N-terminal His-tagged version using pET28a and a compatible helper plasmid that coexpresses minor tRNAs, argU and ileX to compensate for codon usage difference. ORFs 1, 2, and 3 are organized in a sequence reminiscent of that described in E. coli plasmids of the R1 family, cop-tap-rep. ORF6 encoded by IF1, one of the pME2200-specific elements, showed significant similarity to ORF6 encoded by archaeal phage psiM2 of M. marburgensis strain Marburg and may confer the apparent immunity of its host strain ZH3 to infection by phage psiM2. Our data indicate that M. marburgensis plasmids may evolve by a series of gene duplication and excision events.

Adenosine Triphosphatases↗

PubMed1992

Plasmid profile analysis, phage typing, and antibiotic sensitivity of Salmonella dublin from clinical isolates in the United States.

One hundred clinical isolates of Salmonella choleraesuis subsp. choleraesuis serovar dublin (Salmonella dublin) were examined for phage sensitivity, antibiotic resistance patterns, and plasmid content. Computer analysis of the lysis patterns observed by using 27 typing phages divided the S. dublin isolates into 26 groups. One lytic pattern (Designated pattern 16) contained 52% of the isolates examined whereas 16 isolates had unique patterns, and nine patterns had fewer than ten members. Although 14 antibiotic resistance patterns were observed among the 100 isolates, 79% of the isolates grouped in three major patterns. Seven plasmid groups were identified and designated A-G based on the large plasmids found in the isolates. Of the 100 isolates, 28 contained the plasmid profile of Group A, 28 were Group B, 7 were Group C, 34 were Group D, and 1 isolate each was observed in Groups E, F, and G. The strong association between antibiotic resistance pattern and plasmid type suggest that the drug resistance genes are plasmid borne.

Animals↗

PubMed1999

Sequence analysis of plasmid pKJ50 from Bifidobacterium longum.

The complete nucleotide sequence of a plasmid, pKJ50, isolated from an intestinal bacterium, Bifidobacterium longum KJ, has been determined. The plasmid was analysed and found to be 4960 bp in size with a G+C content of 61.7 mol%. Computer analysis of sequence data revealed three major ORFs encoding putative proteins of 31.5 (ORFI), 24.5 (ORFII) and 38.6 kDa (ORFIII). ORFI encodes a protein with a pI of 10.18 and shows relatively high amino acid sequence similarity (more than 60%) with several plasmid replication proteins from Gram-positive and -negative bacteria. Southern blot analysis showed that pKJ50 accumulates an ssDNA intermediate, suggesting that it replicates by a rolling-circle mechanism. Upstream of ORFI, three sets of repeated sequences resembling iteron structures of related plasmids were identified. ORFIII encodes a protein with a pI of 10.97. It also shows a high level of amino acid sequence similarity with some plasmid mobilization proteins. Upstream of ORFIII, a 12 bp stretch resembles an oriT DNA sequence with inverted repeats identical to those found in conjugative plasmids. Hydropathy plot analysis of ORFII, encoding an acidic protein (pI = 4.95), suggests it is a transmembrane protein. Several interesting palindromic sequences, repeat sequences and hairpin-loop structures around ORFI, which might confer regulatory effects on the replication of the plasmid, were also noted. Reverse transcriptase PCR (RT-PCR) and in vitro translation confirmed the expression of ORFI and ORFII. RT-PCR produced amplified DNA fragments of the expected sizes, corresponding to ORFI and ORFII. However, no RT-PCR product corresponding to ORFIII was obtained. In vitro translation showed protein bands of the expected sizes, corresponding to each ORF. A shuttle vector capable of transforming Bifidobacterium animalis MB209 was constructed by cloning pKJ50 and a chloramphenicol resistance gene into pBR322.

Amino Acid Sequence↗

PubMed2003

Bacillus subtilis soil isolates: plasmid replicon analysis and construction of a new theta-replicating vector.

We have searched for plasmids in a collection of 55 Bacillus subtilis strains isolated from various natural sources of the territory of Belarus. Twenty percent of the strains contained one or two plasmids of either 6-8 or approximately 90 kb. Small plasmids were shown to carry a rolling circle replicon of the pC194 type. Four out of the eight large plasmids contained a related theta replicon that has no homolog in databases as shown by sequence determination. A B. subtilis/Escherichia coli shuttle vector based on this replicon was constructed. It has a low copy number (6 units per chromosome) and is stably inherited in B. subtilis. It might thus be a useful tool for DNA cloning. These data extend previous observations, indicating that most of the small plasmids of B. subtilis replicate as rolling circles and belong to the pC194 family. On the contrary, large plasmids appear to form a large pool of theta-replicating determinants, since three different replicons have already been isolated from them.

Amino Acid Sequence↗

PubMed1984

Analysis of plasmids mediating gentamicin resistance in methicillin-resistant Staphylococcus aureus.

Gentamicin-resistance plasmids in methicillin-resistant Staphylococcus aureus isolated from four Australian hospitals have been studied. All the plasmids conferred resistance to gentamicin, tobramycin, kanamycin and all but one conferred resistance to quarternary ammonium compounds. Plasmids which only carried these resistance determinants were ca 15.3 megadaltons and were only found in isolates from one hospital. The most common plasmid was ca 18.0 megadaltons and in addition encoded trimethoprim resistance. Two plasmids, one of ca 19.6 megadaltons and one of ca 28.5 megadaltons were found to carry the penicillinase determinant. However only the larger of these encoded heavy metal ion resistance and was sensitive to quaternary ammonium compounds. EcoR1 analysis indicated that all but the ca 28.5 megadalton plasmid were closely related. The EcoR1 analysis of the ca 28.5 megadalton plasmid indicated that it could have resulted from recombination between a gentamicin resistance determinant and a penicillinase plasmid.

DNA Restriction Enzymes↗

PubMed2006

A first global analysis of plasmid encoded proteins in the ACLAME database.

Many plasmids are mobile genetic elements (MGEs) and, as other members of that group of DNA entities, their genomes display a mosaic and combinatorial structure, making their classification extremely difficult. As other MGEs, plasmids play a major role in horizontal transfer of genetic materials and genome reorganization. Yet, the full impact of such phenomenon on major properties of the host cell, such as pathogenicity, the ability to use new carbon sources or resistance to antibiotics, remains to be fully assessed. More and more complete plasmid genome sequences are available. However, in the absence of standards for storing plasmid sequence data and annotating genes and gene products on sequenced plasmid genomes, the resulting information remains rather limited. Using 503 sequenced plasmids organized in the ACLAME database, we discuss how, by structuring information on the genomes, their host and the proteins they code for, one can gain access to either global or more detailed analysis of the plasmid sequence information, as illustrated by a network representation of the relationships between plasmids.

Bacteria↗

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↗

PubMed1980

Physical and functional mapping of RP4-TOL plasmid recombinants: analysis of insertion and deletion mutants.

Cleavage sites for the restriction endonucleases XhoI, BamHI, HindIII, and EcoRI were mapped on the pTN2 plasmid, a recombinant of TOL and RP4, which specifies the toluene-degrading enzymes in the same way as the wild-type TOL plasmid. The pTN2 plasmid, purified from a strain of Escherichia coli, contained the entire length of the RP4 plasmid (about 54 kilobase pairs [kb]) and the TOL segment (about 56 kb). The TOL segment is inserted at about 12 and 5 kb away from the EcoRI and BamHI cleavage sites of RP4, respectively. Cleavage sites for XhoI, BamHI, HindIII, and EcoRI were also mapped on an insertion mutant, pTN1, and two deletion mutants, pTN81 and pTN9. Analysis of pTN81 and pTN9 allowed estimation of the region of the gene cluster for benzyl alcohol dehydrogenase and benzaldehyde dehydrogenase, as well as the region required for toluate oxygenase activity. Induction of TOL enzymes directed by pTN1 suggested the location and orientation of transcription of the gene cluster for catechol 2,3-oxygenase, 2-hydroxymuconic semialdehyde dehydrogenase, and 2-hydroxymuconic semialdehyde hydrolase. Analysis of strains carrying both pTN9 and a xylR mutant of the TOL plasmid demonstrated that xylR+ is trans dominant over xylR.

Alcohol Oxidoreductases↗

PubMed1996

Genetic analysis of plasmid determinants for microcin J25 production and immunity.

Microcin J25 (MccJ25) is a small peptide antibiotic produced by an Escherichia coli strain isolated from human feces. The genetic determinants for MccJ25 synthesis and immunity have been cloned from the low-copy-number wild-type plasmid pTUC1OO into the compatible vectors pBR322 and pACYC184. Physical and phenotypical analysis of insertion mutations and complementation tests defined three contiguous genes involved in MccJ25 production which span a region of about 2.2 kb. Immunity to the antibiotic is provided by an additional gene adjacent to the production region.

Bacteriocins↗

PubMed1994

[Bacterial genotyping in nosocomial infections].

Hospitalized patients are at unusually high risk of infections, and furthermore, the hospital environment favors the acquisition of resistance to antimicrobial agents, complicating the treatment of nosocomial infections due to drug-resistant pathogens. The prevention of nosocomial infections, based on a surveillance system as an essential element of an infection control program, is the only way to reduce morbidity and mortality. A typing method for strain clonality permits the infection control program to confirm the association between infected patients and the reservoir for the microorganisms of interest and to determine modes of transmission, because the mode of transmission or reservoir may not be the same for multiple strains of a bacterial species. An ideal method of subtyping bacterial isolates from a given species should be simple, rapid, sensitive and discriminatory. Traditionally, once bacterial isolates from an outbreak have been determined to be of the same species, further evaluation for similarity or relatedness has been based on phenotypic methods. Biotyping, serotyping, phagetyping, and antibiotype determination are not always adequately sensitive to distinguish unrelated strains with similar phenotypes. Within phenotypic methods, multilocus enzyme electrophoresis is a powerful tool but because of its complexity it is not likely to become widely available for study of local outbreaks of bacterial infections. In recent years, molecular genetic methods, including plasmid profile, genomic restriction fragment length polymorphism analyses by conventional electrophoresis or by pulsed-field gel electrophoresis, single chromosomal gene polymorphism by DNA hybridization or by PCR amplification, ribotyping, and genomic fingerprinting generated by repetitive element sequence-based polymerase chain reaction, have been useful in evaluating endemic infections and outbreaks of a variety of nosocomial pathogens. For epidemiologic studies, genotyping systems based on defined chromosomal genes or whole DNA polymorphism provide significant advantages over plasmid analysis. Among plasmid-non-based genotypic methods, the choice depends on i) the examination of how much discrimination the method can add for the epidemiologic investigation, ii) the resources available to the laboratory, and iii) the level of expertise of the personnel involved in the testing because, until standardized rules of interpretation are published, the same data may be interpreted in different ways by different investigators. Finally, biotyping, serotyping, and antibiotype determination remain an appropriate first step for the evaluation of apparent outbreaks with the caveat that different strains with the same phenotypic properties may exist concurrently within the same environment. Genotyping may be a second level of analysis to evaluate relatedness of bacterial strains, because the use of molecular biology techniques should support an epidemiologic investigation rather than initiate it.

Bacterial Typing Techniques↗

PubMed1996

Enterohemolytic phenotypes and genotypes of shiga toxin-producing Escherichia coli O111 strains from patients with diarrhea and hemolytic-uremic syndrome.

Thirty-six Shiga toxin-producing Escherichia coli (STEC) O111:H- strains, 18 of which were isolated from patients with hemolytic-uremic syndrome (HUS) and 18 from patients suffering from diarrhea, were investigated for their enterohemolytic phenotypes and genotypes. Twenty-two strains were EHEC hemolysin (EHEC Hly) positive by probe hybridization and by PCR with sequences complementary to the EHEC hlyA gene of E. coli O157:H7, but only 20 of these were hemolytic on blood agar plates. The remaining 14 strains were EHEC Hly negative according to DNA-based methods and did not express the enterohemolytic phenotype. The enterohemolytic phenotype was observed in 16 of 18 (88%) strains from patients with HUS but only in 4 of 18 (22.2%) of the STEC O111:H- strains from patients with diarrhea. All STEC O111:H- strains carried large plasmids, as shown by plasmid analysis, but only plasmids of EHEC Hly probe-positive strains hybridized with the CVD419 probe. A BamHI fragment of approximately 12 kb was cloned from the large plasmid of the E. coli O111:H- strain 78/92 and shown to mediate hemolytic activity when transformed into the E. coli laboratory strain HB101. The EHEC O111 hlyA gene was sequenced completely and shown to have 99.4% sequence identity to the corresponding EHEC O157 hlyA gene of the E. coli O157:H7 strain EDL 933. Our results indicate that detection of EHEC Hly either by DNA-based methods or by investigation of the enterohemolytic phenotype on blood agar alone is insufficient for screening STEC O111 strains. However, the high incidence of EHEC Hly in isolates from patients with HUS and its rare occurrence in isolates from patients with diarrhea may indicate that STEC O111 strains have a distinct pathogenic potential for humans and that the presence of EHEC Hly increases the ability of an STEC O111 strain to cause extraintestinal complications in humans.

Bacterial Toxins↗