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PubMed1984

Plasmid-pattern analysis for the differentiation of infecting from noninfecting Staphylococcus epidermidis.

Isolation of Staphylococcus epidermidis from cultures of blood was differentiated from culture contamination by the detection of identical isolates in two or more consecutive cultures from an infected patient. We used plasmid-pattern analysis as a tool for establishing the identity of individual isolates. In a control study of 15 patients with two or more cultures of blood contaminated with S. epidermidis, analysis revealed that none of the isolates had identical plasmid patterns. This reflected the variety of plasmid patterns among colonizing coagulase-negative staphylococci cultured from skin sites of uninfected patients. In contrast, plasmid-pattern identity was seen among sequential or paired S. epidermidis isolates from a given patient in 32 of 36 patients with documented S. epidermidis infection. The plasmid pattern of each set of isolates from patients was unique. Infections included prosthetic-valve endocarditis in 26 patients, cerebrospinal fluid-shunt or ventriculostomy infections in six patients, intravenous-catheter sepsis in two patients, urinary tract infection in one patient, and osteomyelitis in one patient. Plasmid-pattern analysis may therefore be useful in the diagnosis of S. epidermidis infections.

Anti-Bacterial Agents↗

PubMed1991

Comparison of plasmid profile analysis, antibiogram testing, resistotyping and biotyping in the identification of Escherichia coli isolates from poultry.

Twenty isolates of Escherichia coli from poultry, predominantly from birds with colibacillosis, were collected and plasmid profile analysis, antibiogram testing, resistotyping and biotyping were compared as epidemiological tools for the differentiation and identification of the isolates. Plasmid profile analysis, in conjunction with any one of the other three tests, was found to be more valuable as an epidemiological tool than one test alone.

Animals↗

PubMed1990

Plasmid profile analysis and antimicrobial susceptibility patterns of Shigella isolates from Nigeria.

In an epidemiological survey, plasmid profiles and antimicrobial susceptibility testing of 100 shigella isolates in Lagos, Nigeria was done. All the isolates were sensitive to nalidixic acid, nitrofurantoin and ciprofloxacin. The commonest antimicrobial susceptibility pattern was resistance to ampicillin, colistin sulphate, co-trimoxazole, streptomycin and tetracycline. All but 4 of 100 isolates screened contained one or more plasmids. Plasmid profile analysis distinguished more strains than did antimicrobial susceptibility patterns. A total of 36 isolates was able to transfer resistance plasmids to Escherichia coli K-12 by conjugation. Using in vitro transformation, seven isolates transferred resistance. These plasmids specified resistance to tetracycline, streptomycin, sulphonamide, trimethoprim and ampicillin.

Anti-Bacterial Agents↗

PubMed2001

Molecular subtyping of Shigella flexneri 3a isolates by plasmid profile analysis and pulsed-field gel electrophoresis.

Twenty-two clinical isolates of Shigella flexneri 3a that were obtained from stool specimens from seven outbreaks of infection in northern Taiwan were subtyped and compared using plasmid profile analysis and pulsed-field gel electrophoresis (PFGE). Among 32 isolates, three distinct patterns were found by plasmid profile analysis and six by PFGE, with three different restriction enzymes, XbaI, SfiI, or NotI. Seven PFGE patterns were observed in a comparative analysis using a combination of these three enzymes. Isolates collected from the same outbreaks had the same PFGE combination patterns and were distinguishable from those obtained during other outbreaks. These findings suggest that PFGE may be the most powerful method of genotyping S. flexneri 3a in epidemiological studies.

Electrophoresis, Gel, Pulsed-Field↗

PubMedDate not supplied

Differentiation of Shigella strains by plasmid profile analysis, serotyping and phage typing.

Ninety-one Shigella flexneri and 29 Shigella sonnei strains isolated during 1994 from sporadic cases of shigellosis and healthy carriers were analyzed for plasmid profile in order to compare the discriminating ability of this method with that of serotyping and phage typing. Our study revealed 10 plasmid profiles (PP) among S. sonnei strains. A total of 26 out of 29 (89%) S. sonnei isolates could be placed into two phage types (type 1 and 20) comprising four PP for phage type 1 and seven PP for type 20, respectively. Twenty-three different PP were identified among S. flexneri strains. Each serotype was associated with a specific predominant plasmid profile, except serotype 2a. This serotype, the most frequently isolated in Romania, was still rather homogeneous: 33 out of 39 isolates belonged to phage type 125, 27 of which could be placed into two related PP (F10 and F17). Comparison of plasmid patterns of epidemiologically independent S. flexneri serotype 2a isolates with those exhibited by 45 serotype 2a isolates associated to six independent outbreaks revealed the same homogeneity. Thirty-eight strains, representing 4 of 6 outbreaks, had F10 and F17 plasmid patterns. The discrimination indices (D) for plasmid profile analysis alone (D = 0.890) and for the combination of serotyping and phage typing (D = 0.841) indicate that both typing systems have a nearly similar ability of discriminating among S. flexneri strains. By combining the results of the three typing methods, a total of 42 types are distinguished and the D value is 0.942. Our data suggest that plasmid profile analysis can complement phenotyping methods resulting in a degree of discrimination that cannot be achieved by either system alone.

Bacterial Typing Techniques↗

PubMed1986

Plasmid profile analysis as a tool for characterization of epidemic Shigella sonnei strains.

The usefulness of plasmid profile analysis as a marker to determine the strain number of Shigella sonnei implicated in an epidemic outbreak and its prevalency in our community was studied. We could define two multirresistant strains and determine that the drug resistance was plasmid mediated in both. One strain carried a tra- plasmid which encoded (Sm, Sd)r, the other one carried two tra+ plasmids which encoded (Ap-Cb, Sm, Sd)r and (Sm, Km-Nm, Sd, Tp)r respectively. These strains and some coisolates collected during the later phase of the outbreak carried cryptic plasmids which showed similar electrophoretic mobilities.

DNA, Bacterial↗

PubMedDate not supplied

Plasmid profile analysis and restriction enzyme analysis in characterizing Shigella flexneri isolates from an outbreak.

Shigella flexneri strains which are multiply resistant to antimicrobial agents were isolated from 11 children from an orphanage in Cluj-Napoca during an epidemiological investigation initiated by the Department of Epidemiology. Plasmid profile analysis and restriction endonuclease analysis were used in conjunction with biotyping, serotyping and antimicrobial susceptibility testing for identifying epidemiological related isolates. All strains were serotype 2a and with one exception all of them showed the same resistotype. Plasmid profile analysis differentiated S. flexneri isolate into four patterns, with two common plasmids of 3.5 and 1.9 kb. This study indicates that this outbreak was caused by at least two different strains of S. flexneri which were not differentiated by the classical technique-biotyping, serotyping and antimicrobial susceptibility pattern.

Child, Preschool↗

PubMed2003

Host specificity of mollicutes oriC plasmids: functional analysis of replication origin.

Recently, artificial oriC plasmids containing the chromosomal dnaA gene and surrounding DnaA box sequences were obtained for the mollicutes Spiroplasma citri and Mycoplasma pulmonis. In order to study the specificity of these plasmids among mollicutes, a set of similar oriC plasmids was developed for three mycoplasmas belonging to the mycoides cluster, Mycoplasma mycoides subsp. mycoides LC (MmmLC), M.mycoides subsp. mycoides SC (MmmSC) and Mycoplasma capricolum subsp. capricolum. Mycoplasmas from the mycoides cluster, S.citri and M.pulmonis were used as recipients for transformation experiments by homologous and heterologous oriC plasmids. All five mollicutes were successfully transformed by homologous plasmids, suggesting that the dnaA gene region represents the functional replication origin of the mollicute chromosomes. However, the ability of mollicutes to replicate heterologous oriC plasmids was found to vary noticeably with the species. For example, the oriC plasmid from M.capricolum did not replicate in the closely related species MmmSC and MmmLC. In contrast, plasmids harbouring the oriC from MmmSC, MmmLC and the more distant species S.citri were all found to replicate in M.capricolum. Our results suggest that the cis-elements present in oriC sequences are not the only determinants of this host specificity.

Amino Acid Sequence↗

PubMed1989

Restriction enzyme analysis of plasmid DNA and bacteriophage typing of paired Staphylococcus aureus blood culture isolates.

We compared restriction enzyme analysis of plasmid (REAP) DNA profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of Staphylococcus aureus blood isolates from patients with multiple positive blood cultures. Isolates from 17 pairs did not have detectable plasmids. Isolates from 33 pairs had plasmids classified into 17 distinct REAP DNA profiles. Paired isolates from 31 of these episodes were identical to one another. By phage typing, 35 pairs had strong lytic reactions to a phage(s), 9 pairs lacked strong reactions, and 6 pairs consisted of a strongly reactive isolate and an isolate with no strong reaction to a phage. When consolidated into 11 general phage groups, pairs from 44 of the 50 episodes were in the same general group. REAP DNA profiles were highly reproducible (99%), whereas phage typing was not. REAP DNA profiling is superior to phage typing as a technique for determining similarities and differences among S. aureus blood isolates.

Bacteriophage Typing↗

PubMed1999

Molecular typing of multiresistant Klebsiella pneumoniae isolated from children from northern Jordan.

Twenty-nine clinical isolates of community acquired Klebsiella pneumoniae obtained from 17 children with malnutrition were characterized by antibiotic susceptibility, plasmid analysis, and random amplified polymorphic DNA (RAPD) techniques. Disc diffusion methodology was used to test the susceptibility of the isolates to 13 antibiotics: amoxycillin, cephapirin, ceftazidime, cefoxitin, cefotaxime, aztreonam, gentamicin, ciprofloxacin, chloramphenicol, erythromycin, nalidixic acid, trimethoprim and amoxycillin-clavulanic acid. All the isolates showed multiresistance patterns (15 patterns) ranging from resistance to two antibiotics to resistance to 10 antibiotics. All isolates were resistant to amoxycillin and erythromycin. Ten K. pneumoniae isolates producing extended-spectrum beta-lactamases (ESBLs) as evidenced by the double-disc diffusion synergy test were isolated sporadically from six patients. Six of these 10 isolates were hyperproducers of ESBL, which resulted in increased resistance to the beta-lactam-beta-lactamase inhibitor combination amoxycillin-clavulanic acid. Plasmid analysis showed plasmid ranging in size from 48 kilobases (kb) to 1.4 kb. All the 29 isolates shared the same plasmid 26 kb. There was a consistent relationship between antibiotype and plasmid profiles for each pair of isolates obtained from five individual patients. RAPD analysis using a single (10-mer) primer demonstrated that the isolates that have the same antibiotype and the same plasmid profile had different RAPD fingerprint patterns. These results demonstrate that the RAPD technique is better than antibiotype characterization and a plasmid analysis profile for typing K. pneumoniae as well as for revealing strain differences.

Anti-Bacterial Agents↗

PubMed1982

Plasmid pattern analysis of Staphylococcal epidermidis isolates from patients with prosthetic valve endocarditis.

The electrophoretic pattern formed by individual bacterial plasmid DNA molecules of differing molecular size was evaluated as an epidemiological marker among isolates of Staphylococcus epidermidis from patients with prosthetic valve endocarditis (PVE). Purified covalently closed circular plasmid DNA was obtained from selected isolates, and 79% of the plasmids were found to be less than 10 megadaltons in size; only these small plasmids were sought in subsequent screening gels. Crude cell lysates obtained by a rapid lysis technique and screened by agarose gel electrophoresis revealed the presence of one or more small plasmids in 54 of 58 (93%) PVE isolates; 79% contained two or more. Among 45 plasmid-containing isolates from cases of sporadic PVE at three institutions there were no identical plasmid patterns, although several isolates differed by a single plasmid. In contrast, among nine isolates from a cluster of cases of PVE in Canada, two groups of three isolates each had identical plasmid patterns. Additional clinical data suggested that these isolates were epidemiologically related. Phage typing distinguished one of the groups with plasmid pattern identity, but not the other, from the three isolates with dissimilar patterns. Plasmid pattern analysis shows promise as an epidemiological marker for clinically important isolates of S. epidermidis.

DNA, Circular↗

PubMed1993

Interaction between mitochondrial DNA and mitochondrial plasmids in Claviceps purpurea: analysis of plasmid-homologous sequences upstream of the lrRNA-gene.

Homology of two linear, mitochondrial (mt) Claviceps purpurea plasmids, pClK1 and pClT5, to the upstream region of the large ribosomal RNA gene in the mtDNA of three strains (W3, T5 and K) has been investigated in detail to explore the widespread phenomenon of homology between mt plasmids and mtDNA in C. purpurea. Sequence comparison indicates that recombination between free plasmids and mtDNA is the cause of the observed homology. The process is similar to the integration of the structurally related adenoviruses into the mammalian genome. As in other fungi, palindromic sequences seem to be involved in this mitochondrial recombination process.

Base Sequence↗

PubMed1997

ORF7 of yeast plasmid pGKL2: analysis of gene expression in vivo.

ORF7 of Kluyveromyces lactis killer plasmid pGKL2 (k2) is capable of encoding a putative RNA polymerase subunit of 16 kDa. RNA analysis detected a single, plasmid-dependent ORF7 transcript of 550 nt indicating that the gene is transcribed mono-cistronically. Attempted one-step gene disruption of ORF7 resulted in chromosomal integration of the marker gene rather than the formation of stable recombinant k2ORF7(0) deletion plasmids. Thus, ORF7 appears to be a potential cis-dominant locus the integrity of which is indispensable for plasmid stability. The ORF7 gene product was over-produced as a c-myc-tagged fusion protein in Escherichia coli. Western-blot analysis of total yeast protein extracts using an antibody against this Orf7-c-myc fusion product identified a protein band with an apparent molecular weight of 17 kDa. This protein corresponds in size to the predicted product and is only detectable in plasmid-carrying killer yeasts.

Blotting, Northern↗

PubMed2002

Shifting serotypes, plasmid profile analysis and antimicrobial resistance pattern of shigellae strains isolated from Kolkata, India during 1995-2000.

One hundred and sixty-six shigellae strains, isolated from stool samples of paediatric patients (< 5 years old) at a Childrens' Hospital in Kolkata, India during the period of 1995-2000 were examined for serotyping, drug resistance pattern and plasmid profiles. Sh. flexneri (58 %) was found to be commonest isolate of total shigellae, followed by Sh. sonnei (28 %), Sh. boydii (9%) and Sh. dysenteriae (5%). This profile of species was in sharp contrast to the picture obtained before 1995, when Sh. dysenteriae 1 predominated over Sh. flexneri. In Sh. flexneri strains, Sh. flexneri 2a (35%) was the most prevalent serotype, following Sh. flexneri 3a (31%), Sh. flexneri 6 (14%), Sh. flexneri 2b (11%) and Sh. flexneri 4 (9%). Resistance patterns of the strains to 12 commonly used antimicrobial agents and minimum inhibitory concentrations (MICs) of the antibiotics were also tested. All strains were found uniformly susceptible to norfloxacin, but more than 90% strains were resistant to tetracycline, co-trimoxazole and 67% strains were resistant to ampicillin. Resistance to amoxicillin, chloramphenicol and nalidixic acid was found in 55% (range 45-74%), 46% (range 40-60%) and 29% (range 15-40%) strains respectively. Overall, shigellae strains showed statistically significant increase in resistance against tetracycline, nalidixic acid and furazolidone (P < 0.05) over the years of this study. This indicates decreased efficacy of furazolidone, cotrimoxazole and nalidixic acid for the empirical treatment of shigellosis in Kolkata. Although a few strains showed intermediate susceptibility to ciprofloxacin (4%) and cefotaxime (10%) by disk diffusion test, but the MICs of those antibiotics were within the normal limits. Almost 57% of the strains were resistant to four or more drugs with high MICs of the antibiotics. Plasmid profile analysis revealed presence of large plasmid of 220 kb in majority of the strains except in Sh. sonnei and a correlation between presence of smaller plasmids and shigellae serotypes. Hence this study reports epidemiological change of shigellae species in Kolkata, India with regard to serotypes and antibiotic resistance patterns.

Anti-Bacterial Agents↗

PubMed1983

Pedigree analysis of plasmid segregation in yeast.

We have used pedigree analysis to investigate the mitotic segregation of circular and linear DNA plasmids in Saccharomyces cerevisae. Circular ARS plasmids, which bear putative chromosomal replication origins, have a high segregation frequency and a strong bias to segregate to the mother cell at mitosis. The segregation bias explains how the fraction of plasmid-bearing cells can be small despite the high average copy number of circular ARS plasmids. Linear ARS plasmids do not show strong segregation bias, nor does the 2 mu ori-containing plasmid YEp 13, when it is present in strains containing intact 2 mu circles. In the absence of endogenous 2 mu circles, YEp 13 behaves like an ARS plasmid, showing a strong maternal segregation bias. The presence of a centromere on circular ARS plasmids eliminates segregation bias. We discuss a model for plasmid segregation, which explains these findings and the possible biological significance of mother-daughter segregation bias.

DNA↗

PubMed2006

Characterization of Shigella strains in Iran by plasmid profile analysis and PCR amplification of ipa genes.

To characterize Shigella clinical strains, we studied 82 Shigella strains recovered from 719 stool samples of patients with bloody diarrhea in Shiraz, Iran, over the period from April to October 2003. Serological assay classified the Shigella isolates as follows: 61 (74.39%) Shigella sonnei isolates, 16 (19.51%) Shigella flexneri isolates, 3 (3.65%) Shigella boydii isolates, and 2 (2.43%) Shigella dysenteriae isolates. In an antibiogram test, all Shigella strains were susceptible to ceftazidime, ciprofloxacin, and ceftriaxone. They showed high degrees of sensitivity to nalidixic acid, gentamicin, cephalothin, and amikacin. Approximately 90.24% of the Shigella isolates were resistant to co-trimoxazole. The plasmid profile patterns of all strains were determined by a modified alkaline lysis method. The average number of plasmid bands for each strain was 9.5. By plasmid profile analysis we identified 56 genotypes among all isolates and 42, 14, 3, and 2 genotypes among the S. sonnei, S. flexneri, S. boydii, and S. dysenteriae strains, respectively. PCR assays showed that all isolates were positive for two virulence genes, ipaBCD and ipaH. In conclusion, these data mandate local monitoring of drug resistance and its consideration in the empirical therapy of Shigella infections. These results also demonstrate that plasmid profile analysis is more reliable than antibiotic susceptibility pattern analysis for the identification of Shigella epidemic strains isolated in Iran.

Adolescent↗

PubMed2006

Identification of virulence-associated genes of Pseudomonas viridiflava activated during infection by use of a novel IVET promoter probing plasmid.

Analysis of virulence mechanisms of plant pathogens is often limited by the lack of genetic tools that can be used to identify genes that are preferentially expressed during their interactions with plants. In the present study, we used the newly constructed IVET (in vivo expression technique) plasmid pIviGK and the corresponding antibiotic resistance-based selection method to identify genes that encode pathogenicity factors of the soft rot-causing bacterium Pseudomonas viridiflava. These included pel, the gene encoding pectate lyase, which is responsible for the development of soft rot symptoms. We have also isolated and characterized the gene mviNpv encoding a putative novel membrane associated virulence factor of P. viridiflava. A mutation in mviNpv was shown to influence motility as well as virulence of P. viridiflava. The mviNpv gene is expressed to a moderate level in LB media and its expression increases under inducing conditions as was shown by measuring in planta expression dynamics of the fused gfp reporter gene.

Capsicum↗

PubMed1996

Use of restriction endonuclease analysis of plasmids and pulsed-field gel electrophoresis to investigate outbreaks of methicillin-resistant Staphylococcus aureus infection.

We used restriction endonuclease analysis of plasmids (REAP) and pulsed-field gel electrophoresis (PFGE) to investigate clusterings of methicillin-resistant Staphylococcus aureus (MRSA) infections in our orthopedic unit, neurosurgery unit, intensive care unit, and burn unit. Fourteen different strain types were identified by REAP and 10 different strain types were identified by PFGE among 25 MRSA isolates collected during these incidents of infection. Though neither technique was clearly superior to the other for typing MRSA isolates, REAP is recommended as a relatively simple and reproducible technique for the preliminary investigation of MRSA infection outbreaks in clinical settings.

Bacterial Typing Techniques↗