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PubMed1979

Nucleotide sequence determination of a strong promoter of the colicin E 1 plasmid. Analysis of restriction sites protected by RNA polymerase interactions before and after limited transcription.

This paper presents the location and nucleotide sequence of a strong promoter of ColE 1. This promoter is of interest because of its greatly enhanced activity in the supercoiled state of the plasmid DNA (3) and its possible role in the maintenance of the plasmid replicon (4). This strong promoter is located at the restriction endonuclease Hae III f-h site 0.13 map units from the single EcoR 1 site proximal to the origin of DNA replication. The nucleotide sequence of the Hpa II l fragment of ColE 1 which contains this promoter has been determined. Initiation of transcription at this promoter occurred at two positions. Limited transcription by omitting one of the four nucleotide triphosphates allowed transcription to proceed to the fourth (-UTP) and to the twelfth (-CTP) nucleotides respectively. This was used to probe the interaction between RNA polymerase and the ColE 10.13 promoter by means of restriction cutting at the Hae III site at =27 and the Hha I site at +17. RNA polymerase binding alone blocks restriction cutting at the HAE III site but not at the Hha I site. Limited transcrption to the fourth nucleotide resulted in blocking at both sites. Transcription to the twelfth nucleotide resulted in partial cutting at the Hae III site and blocking at the Hha I site.

Base Sequence↗
PubMed2002

Serotypes, virulence factors, antibiotic sensitivity, beta-lactamase activity and plasmid analysis of Salmonella from children with diarrhea in Tripoli (Libya).

A total of 21 Salmonella strains isolated in Libya (16 from children with diarrhea and 5 from healthy controls) were serotyped and studied for their cell invasive ability, production of cytotoxin, antibiotic susceptibility, beta-lactamase activity and plasmid profiles. Eight different serotypes of Salmonella were identified: 6 S. saintpaul, 4 S. wien (1 from control), 2 S. newport, 2 S. muenchen (1 from control), 2 S. typhimurium (1 from control), 2 S. hadar (1 from control), 2 S. reading (1 from control), 1 S. kottbus. Twenty (95%) were positive in the invasiveness assay using HeLa cells, and all (100%) were negative for cytotoxin production in HT29 cells. More than 40% were resistant to ampicillin, cefalexin, cefamandole, cefoperazone, chloramphenicol, gentamicin, mezlocillin and trimethoprimsulphamethoxazole and 100% were susceptible to the new quinolones. Most (67%) of the strains harbored plasmids and 43% produced beta-lactamase. A strong association was observed between the presence of more than one plasmid, beta-lactamase activity, and multiple-resistance to antimicrobial agents and serotypes S. saintpaul and S. wien. Curing experiments with acridine orange showed that 2 plasmids (33 and 1.4 megadaltons) might be responsible for the resistance to chloramphenicol and gentamicin. The present study demonstrated that multiple-resistant salmonellae are widespread in Libya and the resistance is mainly plasmid mediated.

Anti-Bacterial Agents↗
PubMed1987

Nosocomial clonal dissemination of methicillin-resistant Staphylococcus aureus. Elucidation by plasmid analysis.

An outbreak of methicillin sodium-resistant Staphylococcus aureus (MRSA) infection and colonization, mainly centered in the vascular surgery service, occurred in a 1000-bed tertiary care center between December 1983 and December 1984. Methicillin-resistant S aureus isolated before and during the outbreak was studied by both bacteriophage typing and by restriction endonuclease digestion of bacterial plasmid DNA. Bacteriophage typing was discrepant in nine (56%) of the 17 repeated analyses compared with one (3.4%) of the 29 for plasmid profiling. These typing methods revealed that the epidemic strain was introduced to the hospital from the community 15 months before the outbreak. The outbreak was caused by cross-transmission of the epidemic strain by health care personnel and was controlled by treatment of colonized personnel, education of personnel, and institution of barrier precautions for colonized or infected patients. Plasmid profiling with restriction endonuclease digestion was easier, more rapid, and more specific than bacteriophage typing in the evaluation of this outbreak.

Cross Infection↗
PubMed1985

Investigation by pyrolysis mass spectrometry, phage pattern and plasmid analysis of staphylococci that have reverted from 'L'-phase to bacterial phase.

No major differences have been found in series of Staphylococcus aureus strains which reverted from 'L'-phase, either by pyrolysis mass spectrometry or by phage-typing or sensitivity testing. In 'L'-phase they have been subcultured for a long time or transformed/reverted many times into/from 'L'-phase. Plasmids were lost during transformations/reversions, but there was some difference between the tetracycline-connected plasmids on the one hand and the erythromycin-connected ones on the other.

L Forms↗
PubMed1986

Prevalence of colonization with antibiotic resistant gram-negative bacilli in a nursing home care unit: the importance of cross-colonization as documented by plasmid analysis.

A prevalence study was carried out on a 100-bed Veterans Administration nursing home care unit to determine the extent of colonization with gentamicin-resistant gram-negative bacilli (GRGNB). Hand cultures of 12 employees and 17 environmental cultures were negative. Twenty-six of 86 (30%) patients were colonized with 49 GRGNB. Sixteen patients (19%) had urinary colonization. Multivariate analysis revealed significant associations between rectal or perineal colonization (P less than 0.01), and the presence of a urinary device (82% condom catheters) (P less than 0.05), with urinary colonization. The most common isolates were Providencia stuartii (20), Escherichia coli (nine) and Klebsiella pneumoniae (nine). Twenty-six of 49 isolates carried plasmids. Restriction endonuclease digestion of plasmid DNA was performed for 21. Cross-colonization, as defined by the presence of the identical species with the identical restriction endonuclease digestion profile of purified plasmid DNA found in different patients, was observed for eight of 21 (38%) strains. All were geographically clustered. No strains could transfer gentamicin-resistance by conjugation and only two plasmids could transform our E coli recipient to gentamicin resistance. One E coli plasmid was identical to two Citrobacter freundii plasmids and a P stuartii plasmid isolated from three different patients. This 105 kb plasmid is conjugative and encodes resistance to ampicillin, carbenicillin, tetracycline, and sulfonamides. Thus, 57% of strains were cross-colonizing or contained identical R-plasmids. Southern hybridization using a 1 kb TEM-1 gene probe demonstrated sequences homologous to this probe in five of five nursing home plasmids examined.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1993

Comparison of antimicrobial susceptibility, beta-lactamase production, plasmid analysis and serum bactericidal activity in Edwardsiella tarda, E. ictaluri and E. hoshinae.

Antimicrobial susceptibility profiles of clinical and environmental isolates of Edwardsiella demonstrate that the three species are susceptible to beta-lactam antibiotics. All strains were susceptible to two quinolones tested and to gentamicin and doxycycline. E. tarda and E. hoshinae were resistant to clindamycin, whereas E. ictaluri was moderately susceptible. beta-Lactamase was produced by all strains of E. tarda, but not by E. hoshinae or E. ictaluri. A 54-kb plasmid was detected in six of 13 E. hoshinae strains. Five of the 10 E. tarda isolates studied gave an identical plasmid pattern of four plasmids ranging in size from 76-kb to 5.0-kb. One strain exhibited a 54-kb plasmid; four strains did not contain plasmid DNA. All E. ictaluri isolates contained a 5.7-kb and a 4.9-kb plasmid. E. tarda and E. ictaluri strains were resistant to human serum 20%; 12 of 13 strains of E. hoshinae were also serum resistant. Serum resistance may play an important part in the pathogenicity of these species.

Anti-Bacterial Agents↗
PubMed1985

Mutational resistance as the mechanism of acquired drug resistance to aminoglycosides and antibacterial agents in Mycobacterium fortuitum and Mycobacterium chelonei. Evidence is based on plasmid analysis, mutational frequencies, and aminoglycoside-modifying enzyme assays.

Possible mechanisms of drug resistance of Mycobacterium fortuitum and Mycobacterium chelonei to antibacterial agents were investigated. Single-step mutational frequencies were low (generally less than or equal to 10(-7] for cefoxitin, doxycycline, erythromycin, and sulfamethoxazole and relatively high (10(-4) to 10(-7] for kanamycin and amikacin. Aminoglycoside-susceptible strains of both species contained an aminoglycoside acetyltransferase (3)-III or IV. No additional enzymes were seen with laboratory or clinically acquired aminoglycoside resistance. Plasmids of several sizes were present in some susceptible isolates of both species, but acquired resistance was not associated with a change in the apparent size or number of these plasmids. Isolates with acquired resistance to amikacin were resistant to the other 2-deoxystreptamine aminoglycosides but showed little or no change in minimal inhibitory concentrations to streptomycin, suggesting either a difference in cellular uptake between the 2 groups of drugs or, more likely, different binding sites on the ribosome. In 59 patients treated with 63 courses of therapy with a single agent for 1 month or longer, the development of resistance was observed only twice (3%). Both isolates had high mutational frequencies (10(-4) and 10(-5]. These studies support mutational resistance as the mechanism of acquired resistance to antibacterial agents in M. fortuitum and M. chelonei.

Adolescent↗
PubMed2002

Antimicrobial susceptibility and plasmid analysis of Actinobacillus pleuropneumoniae isolated in Taiwan.

Sixty Actinobacillus pleuropneumoniae (App) strains from pigs in Taiwan were examined. Serotyping revealed that these belonged to serovars 1 (n=53), 2 (n=3), and 5 (n=4). Agar disk diffusion susceptibility testing of the isolates showed 55 (92%) were resistant to three or more antimicrobial agents. Six resistance patterns were observed. Ampicillin-chloramphenicol-flumequine-nalidixic acid-streptomycin-sulfonamide/trimethoprim-tetracycline was the most common multi-resistance pattern. Minimal inhibitory concentration of 14 antimicrobial agents was determined. The isolates were highly susceptible to ceftiofur and trimethoprim in vitro. Isolates were resistant to streptomycin, ampicillin, and nalidixic acid. All isolates were examined for the presence of plasmids using the alkaline lysis method. Forty three (72%) isolates had four plasmid bands with an approximate sizes of 3.5, 4.3, 5.8 and 6.0 kb; 12 (20%) had three bands at 3.5, 4.3 and 5.2 kb, and 5 (8%) had no plasmid bands. Antimicrobial resistance plasmids were detected in resistant strains of App. Three antimicrobial resistance plasmids were transformed into E. coli DH5 alpha. pTMY1 (4.3 kb) encoded a streptomycin kinase and a dihydropteroate synthase; pTMY2 (6.0 kb) encoded ROB-1 beta-lactamase and aminoglycoside 3'-phosphotransferase; pTMY3 (5.2 kb) encoded only ROB-1 beta-lactamase. The 4.3 kb plasmid was sequenced and consisted of 4242 bp with 42.9% GC content. The 4.3 kb plasmid DNA sequence was 98% homologous to a plasmid previously isolated from Pasteurella haemolytica.

Actinobacillus Infections↗
PubMed1994

[Drug resistance and plasmid analysis in Pseudomonas aeruginosa].

One hundred and ninety seven strains of Pseudomonas aeruginosa were isolated from urine (59 strains), wound pus (60 strains), sputum (30 strains), stool (30 strains) and eye discharge (18 strains) at Kaohsiung medical College Hospital. These strains were serotyped with antisera against O antigens and tested with twelve different antimicrobial agents. The results showed that the most frequently isolated strains were serotype E (41.1%), followed by serotype B (20.3%), serotype F (10.7%) and serotype L (9.1%). In in vitro susceptibility testing, all isolated strains were resistant to chloramphenicol and tetracycline. Otherwise, these isolates were highly susceptible to ceftazidime (95.4%), enoxacin (89.3%) and piperacillin (87.8%). The isolates from urine exhibited more multiple drug resistance patterns than those of other specimens. When plasmid content was analysed from pseudomonas aeruginosa, only 15.2% (30/197) of isolates carried plasmids. By conjugation, transformation and mobilization experiments, it was shown that 13.3% (4/30) of plasmid carrying strains contained R plasmids.

Chloramphenicol↗
PubMed1990

Plasmid analysis and restriction fragment length polymorphisms of chromosomal DNA allow a distinction between Borrelia burgdorferi strains.

We examined the relationships of the genomes of five strains of Borrelia burgdorferi isolated from ticks, two from North America, including the type strain B31, and three from Switzerland. We determined restriction fragment length polymorphisms by using eight cloned DNA fragments as hybridization probes to genomic Southern blots. Two divergent patterns were observed, represented by B31 and one Swiss strain on the one hand and the two other Swiss strains on the other. The second American strain resembled B31. One of the DNA probes allowed distinction between the closely related strains within a group. The close resemblance of one Swiss strain to the North American strains suggests the possibility of their European origin. All five strains carried a circular plasmid of about 29 kb, and three contained an additional species of about 9 kb, both of which exhibited homology between the strains. The profiles of linear plasmids revealing species of 5.1 kb to 58 kb reflected the polymorphisms of chromosomal DNAs. Linear plasmids of similar size shared DNA sequence homology. Some of the smaller plasmids tended to become lost during cultivation.

Animals↗
PubMed1994

Antimicrobial susceptibility and plasmid analysis of Campylobacter jejuni isolated from diarrhoeal patients and healthy chickens in northern India.

Seventy-five strains of Campylobacter jejuni isolated from humans with diarrhoea (45 strains) and healthy chickens (30 strains) were tested for their susceptibility to different antimicrobial agents: ampicillin, tetracycline, erythromycin, gentamicin, kanamycin, furazolidine and quinolones (nalidixic acid, norfloxacin, ciprofloxacin). The frequencies of resistance to ampicillin and tetracycline were 16 and 9.3% respectively. Two strains (2.7%) exhibited resistance to quinolones as mentioned. One strain (1.3%) was resistant to erythromycin, and both ampicillin plus tetracycline. One strain (1.3%) exhibited resistance to multi-drugs (ampicillin, tetracycline and erythromycin). Resistance to ampicillin was higher in human strain (22.2%) compared to chickens (6.7%). On the contrary, the frequency of resistance to tetracycline was higher in chicken strains (13.3%) than in human (6.7%). All the ampicillin-resistant strains produced beta-lactamase. None of the ampicillin, erythromycin and quinolone-resistant strains contained any plasmid but all the tetracycline-resistant strains contained 23 kilobase (kb) plasmid which could be transferred to an ampicillin-resistant C. jejuni strain. This study thus shows that ampicillin and tetracycline resistance in C. jejuni are common in northern India. Ampicillin resistance is chromosomally determined but tetracycline resistance is mediated through 23 kb plasmid.

Animals↗
PubMed1989

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

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

Animals↗