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A mathematical model for lambda dv plasmid replication: analysis of wild-type plasmid.

A mathematical model for lambda dv plasmid replication in a growing single cell of Escherichia coli has been formulated and solved numerically. Quantitative description of the molecular control mechanism for initiation of lambda dv replication presumes regulatory functions of repressor and initiator proteins and transcriptional activation of the origin region. Random selection of a single plasmid for activation and replication is assumed, as is regular plasmid segregation to daughter cells. The model is capable of simulating the periodic changes in each regulatory element and the plasmid copy number during the cell cycle. The calculated average copy number, repressor concentration, and timing of plasmid replication agree well with experimental data. The simulated lambda dv plasmid replication rate is controlled primarily by transcription frequency. Initiation of plasmid replication is not related to variations in the levels of repressor or initiator proteins during the cell cycle. Simulation studies of perturbations in plasmid and repressor segregation indicate that replication regulation of the lambda dv plasmid compensates to readjust copy number to normal values in a few generations. Implications of these studies relative to the molecular mechanisms of replication control are discussed.

DNA Replication↗

Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed.

Within the biopharmaceutical industry, recombinant plasmid DNA is used both as a raw material (e.g. in lentiviral and AAV vector production) as well as an active ingredient (e.g. in DNA vaccines). Consequently, many analytical laboratories are routinely involved with plasmid DNA topoisoform qualitative analysis and quantification. In order to reliably determine plasmid topology, one must ensure that the methodology employed can reliably, precisely and accurately measure qualitatively and quantitatively all topological isoforms. Presented here are an anion-exchange high-performance liquid chromatography (AEC) and an agarose gel electrophoresis (AGE)-based method developed for this purpose. The strategies undertaken to overcome the respective typical problems of limited linear range of quantitation (for AGE) and isoform resolution (for AEC) are described. Also presented is a subsequent direct comparison (for assay precision/accuracy) of these two methods, as well as a package of species characterization [by chloroquine-AGE, enzymatic digestion, multi-angle laser light-scattering (MALLS) and electron microscopy] undertaken to confirm the identity of a minor supercoiled dimeric concatamer observed by both approaches.

Chromatography, High Pressure Liquid↗

Characterization of endemic strains of Shigella sonnei by use of plasmid DNA analysis and pulsed-field gel electrophoresis to detect patterns of transmission.

Shigellosis is hyperendemic in Utah. Most isolates are Shigella sonnei, making it difficult to identify epidemiologic clustering. To better define transmission, molecular markers and epidemiologic data were examined for 90 cases. Plasmid analysis and pulsed-field gel electrophoresis (PFGE) of the S. sonnei isolates identified 11 and 4 patterns, respectively. Plasmid pattern I infections occurred in 8 day care centers over a 6-month period, suggesting spread between centers. Plasmid pattern III was isolated from children at 3 additional centers and pattern IV was associated with another day care center, suggesting different outbreaks. By PFGE, plasmid groups I and XI appeared identical, as were plasmid groups II and V; plasmid group X had a unique pattern. Plasmid groups III, IV, and VII-IX were closely related PFGE subtypes. Both plasmid analysis and PFGE allow better characterization of S. sonnei transmission patterns of "endemic" strains and could lead to improved control measures.

DNA, Bacterial↗

Restriction endonuclease analysis of plasmid DNA from methicillin-resistant Staphylococcus aureus: clinical application over a three-year period.

OBJECTIVE: To investigate trends in plasmid types of methicillin-resistant Staphylococcus aureus over a 3-year period and determine the clinical impact of plasmid typing. DESIGN: Restriction endonuclease analysis of plasmid DNA (REAP) was used to type 270 clinical isolates. SETTING: A Veterans Affairs Medical Center. RESULTS: Forty-four different REAP types were identified and, of these, 23 appeared only once. Types designed as V1 and V2 were more commonly found in nosocomial cases than in community-acquired cases (p < .05); whereas the K and D types were more common in isolates from community-acquired cases (p < .05). In 30 (11%) isolates, no plasmid was detected. When combined with epidemiological data, REAP typing revealed 4 small outbreaks that would have been missed using traditional epidemiological methods alone. In large outbreaks involving 10 or more cases, REAP typing data revealed unsuspected patterns of transmission. CONCLUSIONS: Multiple plasmid types were present in this endemic setting. Restriction endonuclease analysis of plasmid DNA was a practical and valuable adjunct to traditional epidemiological methods.

DNA Restriction Enzymes↗

Plasmid profile analysis of salmonellae in a large-animal hospital.

The plasmid profiles of salmonellae isolated from the patients and environment of the Purdue Large Animal Hospital were used as markers to identify strains and to assess the impact of improvements in hospital operation on nosocomial salmonellosis. Plasmid profile analysis proved to be more sensitive than either serotyping or antimicrobial susceptibility testing in identifying Salmonella isolates. During June and July 1983, 29 of 34 salmonellae isolated were one of three strains with distinct plasmid profiles: one S. typhimurium var. Copenhagen, and two S. muenchen. Each of these strains was isolated from at least one patient and two environmental sites, suggesting the possibility that infections were hospital-acquired. Patient and environmental sampling was repeated in June and July 1984, after improvements had been made in hospital traffic flow and sanitation. In contrast to 1983, only seven isolates, representing six strains not seen previously, were obtained in 1984. None of these strains was isolated from both patient and environmental sources. The results indicate that the high incidence of clinical salmonellosis in 1983 was largely due to nosocomial infections. The decrease in the incidence of salmonellosis and the absence of the 1983 strains from samples taken in 1984 were presumed to be due to improvements made in hospital operation. This study demonstrates the value of plasmid analysis in monitoring nosocomial salmonellosis in a veterinary hospital.

Animals↗

Characterization of plasmids in aminocyclitol-resistant Staphylococcus aureus: electron microscopic and restriction endonuclease analysis.

Plasmid species isolated from aminocyclitol-resistant Staphylococcus aureus have been analyzed by restriction endonuclease digestion and electron microscopy. These plasmids can be divided into two interrelated groups; intergroup variability is due to the gain or loss of defined DNA sequences. Plasmids pSJ1 and pSJ24 are related to staphylococcal penicillinase plasmid pI524 which was first described over 20 years ago. Both pSJ1 and pSJ24 differ from pI524 by the acquisition of 8 and 4 kbp, respectively, and encode additional resistance to the antibiotics erythromycin and kanamycin. The gain of these resistance determinants suggests that the evolution of staphylococcal resistance plasmids parallels that observed for plasmids of gram-negative bacteria and has serious implications for the spread of antibiotic resistance among the staphylococci.

Aminoglycosides↗

Strain typing among enterococci isolated from home-made Pecorino Sardo cheese.

Three molecular techniques (RAPD-polymerase chain reaction analysis, plasmid profile and pulsed-field gel electrophoresis) were used for a preliminary approach to type, at strain level, enterococci isolated from a 24-h-old home-made Pecorino Sardo (protected designation of origin) cheese. A high genetic polymorphism was found. Clusters obtained by the RAPD technique and plasmid profile analysis contained different strains. Pulsed-field gel electrophoresis proved to be an efficient and highly reproducible typing method. In addition, by combining the results from plasmid profile analysis and pulsed-field gel electrophoresis, it was possible to identify closely related strains probably belonging to the same clonal lineage.

Bacterial Typing Techniques↗

Plasmid profiles of Acinetobacter and Enterobacter species of hospital origin: restriction endonuclease analysis of plasmid DNA and transformation of Escherichia coli by R plasmids.

A total of 37 multi-resistant strains (20 Acinetobacter calcoaceticus and 17 Enterobacter cloacae) were isolated from patients of the Intensive Care Units. All the isolates were examined for resistance to a battery of antimicrobial agents by the disk diffusion method. Plasmid profiles and restriction endonuclease analysis of plasmid DNA by EcoR1 revealed the spread of one A. calcoaceticus and two E. cloacae endemic strains. Transformation experiments on Escherichia coli competent cells by three plasmids established the presence of R plasmids in the multi-resistant isolates.

Acinetobacter↗

Plasmid profile analysis of Salmonella ser. Typhimurium phage untypable strains encountered in diverse geographic regions of India.

Plasmid profile analysis and antibiotic resistance pattern determination were carried out for 117 phage untypable S. Typhimurium strains. Majority of the strains (82%) were resistant to all the seven antibiotics tested, R-pattern being ApCmKmSmSuTcTp, rest (12%) showed heterogenous R-patterns. Plasmid DNA analysis revealed phage untypable strains to harbour large (58.8-114.3 MDal), intermediate size (36 MDal, 42 MDal) and small (1.8-5.2 MDal) plasmids with varying molecular weights. All the phage untypable strains could be subgrouped by plasmid profile analysis into 23 plasmid patterns. Plasmid profile analysis could discriminate large number of phage untypable strains on the basis of their plasmid pattern.

Anti-Bacterial Agents↗

Antibiogram, biotyping and plasmid profile analysis of Acinetobacter calcoaceticus var. anitratus.

Antibiogram, biotyping, and plasmid profile analysis were studied on 115 isolates (40 nosocomial and 75 community strains) of Acinetobacter calcoaceticus var. anitratus isolated from clinical specimens. By disk diffusion method, we found 14 antimicrobial susceptibility patterns. Among them, the gentamicin-resistant patterns were recovered more frequently from nosocomial origin than from community-acquired origin. Fifteen patterns of biocodes were found by using GNI card of AutoMicrobic System (Vitek System). By using rapid alkaline extraction method and agarose gel electrophoresis, 102 isolates (88.7%) contained plasmids, and 72 distinct profiles were found. No particular plasmid profile correlates with specific antibiogram or biotype. However, multiple isolates with identical antibiogram and biotype recovered from the same patient were usually of identical plasmid profile. In view of the heterogeneity of the plasmid patterns that were found, the combined use of antibiogram, biotyping, and plasmid profile analysis could be satisfactory for epidemiological investigation.

Acinetobacter↗

Plasmid DNA analysis, biotyping, and antimicrobic susceptibility as subtyping tests for Klebsiella pneumoniae and Klebsiella oxytoca.

We compared plasmid DNA analysis, biotyping by Vitek, and disk diffusion antimicrobic susceptibility as subtyping tests of Klebsiella pneumoniae and Klebsiella oxytoca. The 92 tested isolates were from alternate, culture-positive patients over 6 months. No outbreak or cluster of infections was recognized during this interval. Plasmid DNA was detected in 85% of the isolates. Each isolate except one had a reproducible absence of plasmid DNA or a reproducible plasmid DNA profile on repetitive testing. Restriction endonuclease enzyme analysis of plasmid DNA was necessary to distinguish differences among some isolates that had only large plasmids. Isolates with only large plasmids represented 18% of the collection. Of the 78 isolates with plasmid DNA, all but two were considered different from one another by plasmid DNA analysis. Biotyping and antimicrobic susceptibility testing were not highly reproducible. In addition, biotyping did not demonstrate a sufficient variety of patterns among the isolates for subtyping purposes. We conclude that plasmid DNA analysis is very useful as a subtyping test for isolates of K. pneumoniae and K. oxytoca. Neither biotyping nor antimicrobial susceptibility as performed in our laboratory had sufficient discriminatory power and reproducibility for subtyping these organisms.

Anti-Bacterial Agents↗

Epidemiologic investigation of nosocomial outbreak of methicillin-resistant Staphylococcus aureus by plasmid pattern analysis.

Nosocomial infections caused by methicillin-resistant Staphylococcus aureus (MRSA) have become an important problem with increasing frequency. In order to learn if plasmid pattern analysis can be used in epidemiologic investigations of MRSA infections, the authors did plasmid extractions of 70 MRSA stock isolates using a rapid lysostaphin lysis method. All isolates carried at least one plasmid. Most of the isolates had one large plasmid of 24-28 megadaltons (Md). Many also carried one or two small plasmids. Accordingly, 12 different patterns were identified. From these background results, we applied this method to the investigation of two small nosocomial outbreaks of MRSA infection. It was found that the analysis of plasmid pattern and restriction endonuclease analysis are more discriminative than antibiograms. Strains with the same antibiograms can be different by plasmid analysis. It is concluded that the plasmid pattern with a restriction endonuclease analysis study is a reliable method for epidemiologic study of MRSA infections.

Cross Infection↗

Comparison of plasmid profile analysis, phage typing, and antimicrobial susceptibility testing in characterizing Salmonella typhimurium isolates from outbreaks.

We compared the phage types, antimicrobial resistance patterns, and plasmid profiles of 20 groups of isolates received at the Centers for Disease Control from Salmonella typhimurium outbreaks between 1975 and 1982 to determine the most useful laboratory method for identifying epidemiologically related isolates of S. typhimurium. In 18 (90%) of the 20 outbreaks, epidemiologically related isolates were identified as being the same by each of the three methods. In a subgroup of nine outbreaks in which isolates unrelated to the outbreak were submitted for comparison, outbreak isolates were differentiated from such control isolates six times (67%) by phage typing alone, four times (44%) by antimicrobial susceptibility testing alone, and eight times (89%) by plasmid profile analysis alone. Epidemic isolates were multiply susceptible, nontypable, or without plasmids in 14 (70%), 1 (5%), and 3 (15%), respectively, of the 20 outbreaks. Plasmid analysis appeared to be at least as specific as phage typing in identifying epidemiologically related isolates of S. typhimurium as being the same or in differentiating them from control specimens; both techniques appeared to be superior to antimicrobial susceptibility testing.

Anti-Bacterial Agents↗

Plasmid pattern analysis of natural bacterial isolates and its epidemiological implication.

Natural isolates of Escherichia coli, Salmonella typhimurium, Klebsiella pneumoniae, and Providencia stuartii were analysed to determine their plasmid content. This data allowed the identification of nosocomial strains of K. pneumoniae and P. stuartii and helped in the differentiation of epidemic strains of E. coli 0111 and S. typhimurium. Phenotypically similar isolates of S. typhimurium could be shown to be of independent origin using plasmid pattern analysis. The dissemination of a particular plasmid through different strains of S. typhimurium resulted in a simulation of a very widely distributed epidemic strain, because the plasmid interfered with the phage type of its host strain in addition to determining resistance properties. Plasmid pattern analysis disclosed two independently existing but interacting epidemic processes: a bacterial 'epidemic' strain may become disseminated over a large territory and may predominate there for a long time; a single plasmid, however, may also become distributed through many different bacterial strains and may spread over a large territory. Plasmid pattern analysis provides a valuable and universal epidemiological laboratory method.

Anti-Bacterial Agents↗

Plasmid-pattern analysis in the differentiation of Staphylococcus epidermidis isolates.

Plasmids carried by 29 Staphylococcus epidermidis isolates from two different origins (invasive and carrier, non-hospital-associated strains) were analysed by agarose gel electrophoresis in order to evaluate any association with virulence. Plasmid patterns were found to be characteristic of individual strains and no particular molecular size band was found to correlate with either of the two categories of isolates. It is concluded that the plasmid pattern analysis carried out could not differentiate between carrier and invasive S. epidermidis, but did provide a useful tool for epidemiological investigation.

Electrophoresis, Agar Gel↗

An outbreak of multiply resistant Pseudomonas aeruginosa in a neonatal unit: plasmid pattern analysis.

An outbreak of infection due to multiply resistant Pseudomonas aeruginosa occurred from March to April 1986 in a neonatal unit. Affected neonates were receiving ventilation support and the mortality rate was high. Plasmid analysis and antibiograms indicated that the outbreak was due to a single strain. A survey of bacteria isolated from respirators, potable water and hands of personnel working in the unit failed to recover the outbreak strain. Lack of sterilization of respirators and overcrowding were considered to be the causes of the outbreak and reinforcement of the importance of aseptic techniques helped in its termination.

Argentina↗

[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↗

Analysis of genetic diversity among Staphylococcus aureus isolates from patients with deep-seated and superficial staphylococcal infections using pulsed-field gel electrophoresis.

Molecular typing was performed to reveal the genetic diversity among Staphylococcus aureus strains causing deep-seated versus superficial staphylococcal infections. Pulsed-field gel electrophoresis with cluster analysis, plasmid and antimicrobial susceptibility profiling of 50 S. aureus strains collected from these 2 groups of patients were undertaken. A total of 19 (designated A through S) distinct genotypes were identified by PFGE of Sma I-digested genomic DNA. The most prevalent PFGE type was L, which accounted for 30% of isolates and was detected among superficial isolates only. The second most prevalent PFGE type, type A (18%), was predominant among deep-seated isolates. Remaining PFGE types varied in distribution between the 2 groups. Plasmid profile analysis revealed that deep isolates harbour plasmids more frequently (comprising 64% of isolates) than superficial isolates (4%) and showed 10 and 2 distinct patterns, respectively, with pattern 1 being the dominant among deep isolates. Antimicrobial susceptibility data suggested an increased prevalence of antibiotic resistance among deep isolates with the majority (40%) exhibiting identical antibiograms compared to superficial isolates. No resistance was detected against clindamycin and vancomycin. The results of our study indicate a previously unrecognized dichotomy of S. aureus strains, causing deep-seated and superficial infections.

Adolescent↗