Megadalton Plasmid Research Articles

Antimicrobial resistance pattern and plasmid profile of Salmonella typhi isolated from an outbreak in Tehran province.

The antimicrobial resistance patterns and plasmid profiles of Salmonella typhi isolates from sporadic cases (n = 33) and an outbreak (n = 48) were compared. Of 28 sporadic drug-resistant isolates, 24 (85.7%) were multiply resistant. The predominant antibiotic resistance pattern was TeCmSmSxTAp, which was also the most common pattern of the outbreak isolates. 13 drug-resistant strains isolated before the outbreak (46.4%) were able to transfer the whole resistance pattern or part of it to Escherichia coli K 12 by conjugation. Although 20 of the sporadic strains contained plasmid DNA, transferable R plasmids were only detected in 13 (65%) of them. Among the outbreak strains, the rate of R plasmid transfer was 92.3%, with only the TeCmSmSxTAp pattern transferred. Plasmid profiling and Hind III endonuclease digestion of plasmid DNA identified a 91.2 megadaltons (Mda) plasmid that was recovered from most of the outbreak isolates and from 4 strains collected before the outbreak. This plasmid coded for TeCmSmSxtAp and transferred the pattern of resistance in toto. The results indicate multidrug-resistant S. typhi as a potential cause of infection in the region.

Plasmid profiles of Klebsiella pneumoniae isolated from horses.

Plasmid profiles of Klebsiella pneumoniae isolated from horses were examined. Thirty-nine strains of K. pneumoniae capsular type 1 (K1) isolated from cervical swabs of mares suffering from metritis, and from semen of stallions showed similar plasmid profile patterns, and all strains possessed a 125 megadaltons (Md) plasmid. There was no difference in plasmid profiles between the heavily-encapsulated and the less heavily-encapsulated strains of K. pneumoniae K1. Non-capsulated variants derived from the strains of K1 showed the same plasmid profile pattern as the parent strains. Plasmid profiles of K. pneumoniae other than K1 were various, and none of these strains possessed the 125 Md plasmid.

Cotransformation of lactococcin-producing, 2.0-mega dalton and erythromycin-resistant pGB 301 plasmids toLactococcus lactis subsp.lactis protoplast

Protoplasts of plasmid-freeLactococcus lactis subsp.lactis LM 0230 and PC4 strains were cotransformed successfully with the plasmid pools ofL. lactis subsp.lactis 484, a lactosefermenting (Lac+), lactococcin-producing (Lap+), lactococcin-resistant (Lapr), sucrosefermenting (Suc+) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Eryr) at the frequencies of 104 transformants/μg of DNA. PC4 protoplasts were transformed at slightly lower frequencies that LM 0230 protoplasts when the same plasmid combinations were used for transformation. Agarose gel electrophoresis of plasmids from three groups of transformants, namely, Lac−Lap−Eryr, Lac+Suc+Lap+LaprEryr, and Lac−Suc+Lap+ LaprLapr, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc+Lap+Lapr and Lac+ phenotypes respectively. The protoplasts could be transformed with low-molecular-weight 2.0 MDa Lap plasmid at a relatively higher frequency than those with high-molecular-weight 65.0 MDa Lac plasmid. All the transformants resembled parent culture 484 in terms of lactic acid production (0.810–0.840%), milk curdling time (6 h), and lactococcin activity (7–12 mm, zone of inhibition) againstListeria monocytogenes, Salmonella typhi, andStaphylococcus aureus. The plasmids and their respective phenotypes in PC4 transformants were genetically more stable than those of LM 0230 protoplasts. The marker plasmid pGB 301 disappeared more frequently from the transformants when present in association with the lowmolecular-weight, high-copy-number 2.0 MDa plasmid, thereby suggesting the incompatibility of these two plasmids.

A conjugative plasmid pTE195 coding for drug resistance and virulence phenotypes from Salmonella naestved strain of calf origin

Salmonella naestved strain AHI-195, of calf origin, harbors a conjugative 95 megadalton (MDa) plasmid, pTE195, which encodes resistance to tetracycline and chloramphenicol and belongs to incompatibility group FII. Moreover, DNA homology between pTE195 and the Salmonella dublin virulence plasmid pTE800 was revealed by digestion with several restriction endonucleases and confirmed by hybridization with different specific probes. These results indicate that pTE195 carries not only genes for drug resistance but also genes for virulence phenotypes such as serum resistance and mouse lethality.

Biological characters of the clinically isolated strains of Escherichia coli O157:H7 in Canada

Biological characters of enterohemorrhagic Escherichia coli (EHEC) strains isolated from food poisoning cases in Canada, 1987-1988 were examined in comparison with that isolated in Saitama, 1990. The Saitama strain was found to belong to biotype I, and biotypes I and II were found in Canada. Most of the strains including the Saitama strain were found to have siderophilins suggesting their advantageous growth in vivo. An enteroinvasive strain with 140 megadalton (Md) plasmid and two colicinogenic strains with 4.8 Md plasmids were detected. All strains were found to have 60 Md plasmids. This plasmid might be specific for this serotype. A few strains were resistant to streptomycin (SM) and/or tetracycline (TC), and a strain transferred its SM-TC resistances by conjugation. Many strains including the Saitama strain were found to produce vero cytotoxin (VT) types 1 and 2 (VT1 and VT2). A few type 2 and untypable toxin producing strains were also detected. VT titers produced by untypable toxin producing strains were over 10 times lower than those by other strains. High annealing temperature for PCR amplification of VT1 gene and a variety of annealing temperatures of VT2 gene suggested that the nucleotide sequences for VT1 gene were well preserved, but that those for VT2 might have some mutations.

The role of 36 megadalton plasmid of Salmonella enteritidis for the pathogenesis in mice.

The pathogenesis associated with the 36 Megadalton (Md) plasmid of Salmonella choleraesuis subsp. choleraesuis serovar Enteritidis (S. Enteritidis) was assessed by using the plasmid-containing strain AL1190, plasmid-cured strain AL1192, and plasmid-reintroduced strain AL1193. After oral inoculation of strain AL1190 or AL1192 to C57BL/6 mice, mesenteric lymph nodes, spleens, and livers were examined for the numbers of viable bacteria and for the histopathological changes. The numbers of bacteria were greater and histopathological changes were severer in these organs of mice inoculated with strain AL1190, than in those with strain AL1192. Strains AL1190, AL1192, and AL1193 showed the equivalent survival rate to sera of guinea pig, calf, and pig, and grew equally well under the iron-limiting conditions. These results suggested that the 36 Md plasmid of S. Enteritidis contribute to the spread of the infection beyond small intestines to mesenteric lymph nodes, spleens, and livers, but not to serum resistance or acquisition of iron.

Epidemiology of penicillin resistant Neisseria gonorrhoeae.

To study the epidemiology of Neisseria gonorrhoeae that exhibit both chromosomal and plasmid-mediated resistance to penicillin. A total of 1589 strains of N gonorrhoeae isolated from patients attending St Mary's Hospital, London were tested for both their susceptibility to penicillin and for their auxotype and serotype. Of the 940 non-penicillinase producing N gonorrhoeae, 840 were considered penicillin sensitive (MIC less than or equal to 0.5 mg/l) and 100 were chromosomally-mediated resistant N gonorrhoeae (CMRNG), (MIC greater than or equal to 1.0 mg/l). Of the 649 penicillinase producing N gonorrhoeae (PPNG), 429 carried the 4.4 megadalton (MDa) penicillinase encoding plasmid and 220 carried the 3.2 MDa plasmid. CMRNG were predominantly serogroup IB (90%). PPNG with 3.2 MDa plasmid were the only group more often serogroup IA (58%) than IB (42%). Serovar IA-1/2 and requirement for arginine, hypoxanthine and uracil (AHU) were associated with increased susceptibility to penicillin whereas serovar IB-5/7 was associated with decreased susceptibility in nonPPNG. There was a significant difference in the distribution of the IA and IB serovars between PPNG carrying either the 4.4 MDa or 3.2 MDa plasmid. AHU and PAOU requiring strains were not found among PPNG and were uncommon among CMRNG. Some clear associations have been found but the pattern among PPNG appears more complex and in most instances could be related to clusters of a single strain over a short time span.

HeLa cell invasion by a strain of enteropathogenic Escherichia coli that lacks the O‐antigenic polysaccharide

The interaction with HeLa cells of an enteropathogenic Escherichia coli (EPEC) strain and its plasmid-cured derivative strain was examined. An O111:NM EPEC strain B171 harbours a 54 megadalton plasmid (pYR111) necessary for the expression of both localized adherence (LA) to HeLa cells and the O-repeating side chain of the lipopolysaccharide. Under light microscopy, the plasmid-cured derivative strain B171-4 was observed to interact with HeLa cells in a pattern distinct from LA. Transmission electron microscopy showed that the bacteria were internalized by HeLa cells. In contrast, strain B171 induced pedestal-like projections and invaginations of the plasma membrane, but was never completely internalized. A quantitative assay to determine the number of internalized bacteria revealed that strain B171-4 was internalized at levels 30-70-fold higher than those of avirulent E. coli strains. Cytochalasin B reduced the levels of internalization of both strain B171-4 and an enteroinvasive E. coli strain (E11), but did not affect LA by strain B171. These results suggest that EPEC strain B171 may carry a specific chromosomally determined surface factor needed to initiate internalization by HeLa cells. However, a plasmid-determined factor alters the nature of this interaction; the combined effects of the chromosomal and plasmid determinants lead to the characteristic attachment of the bacteria in clusters on the surface of the eukaryotic cell.

Molecular characterization of trimethoprim resistance inShigella sonneiin Sicily

During the 3-year period 1985-7, all strains of Shigella sonnei isolated in Catania, Sicily, showed a high level of resistance to trimethoprim (Tp) which was invariably associated with resistance to other antibiotics. Plasmid analysis showed 18 different electropherotypes: 35 of 37 strains harboured a plasmid of 70 Megadaltons (MDa), and 29 of 37 strains a plasmid of 130 MDa. Restriction endonuclease fingerprinting of purified 70 MDa plasmid DNA from different strains demonstrated that these plasmids were similar but not identical. In some strains with transferable Tp resistance, DNA hybridization analysis demonstrated the presence of gene coding for the production of dihydrofolate reductase (DHFR) type V. In contrast, there was no detectable hybridization with DNA probes specific for genes coding for DHFR types I, II and IV. This is the first report of the DHFR type V gene outside Sri Lanka.

Plasmid profiles of antibiotic-resistantShigella dysenteriaetypes 2, 3, 4, 6 and 7 isolated in Ethiopia during 1976–85

Plasmid profile analysis by agarose gel electrophoresis was carried out on 37 drug-resistant strains of Shigella dysenteriae types 2, 3, 4, 6 and 7. These strains were collected between 1976 and 1985 in Addis Ababa, Ethiopia. The plasmid profile of S. dysenteriae type 2 strains with R-type CSSuT did not show middle-sized plasmids likely to code for CSSuT resistance. All strains contained a large plasmid of about 120 megadaltons (MDa), and a cryptic plasmid of about 2.2 MDa. The plasmid profiles of S. dysenteriae type 3 with R-types ACSSuT, SSuT and SSu showed a 4.2 MDa SSu-determinant, which was demonstrated in Escherichia coli K12 recipients resulting from triparental crosses. The ACT determinant in S. dysenteriae type 3 with R-type ACSSuT is probably chromosomally mediated. Cryptic plasmids of about 3.0 and 2.2 MDa were found in all S. dysenteriae type 3 isolates. The 4.2 MDa plasmid featured prominently in the plasmid profiles of S. dysenteriae types 4, 6 and 7 with R-types SSuT and SSu. However, this plasmid was not mobilizable by triparental crosses. There was a relative paucity of transferable plasmids in non-Shiga bacillus isolates. However, incompatibility group N plasmids, coding for tetracycline resistance, were detected.

The invasiveness of different strains of Salmonella enteritidis phage type 4 for young chickens

Five strains of Salmonella enteritidis phage type 4 (PT4) isolated in 1978, 1984 and 1988 were examined for their ability to colonise the caecum and invade the liver of day-old chickens. All strains were capable of caecal colonisation and there were no differences in their colonisation ability in this respect. In contrast there was a gradation in the ability of strains to invade the liver, with strains isolated in 1988 proving the most invasive. Absence of a 38 megadalton (Md) plasmid, which has been shown to be involved in the virulence of S. enteritidis PT4 for BALBc mice, had little effect on the ability of strains of this phage type to colonise the caecum or invade the liver of day-old chickens. These results suggest that recent isolates of PT4 may have enhanced virulence for chickens which is not necessarily associated with the carriage of a 38 Md plasmid.

Increase of the Relative Frequency of Penicillinase-producing Neisseria gonorrhoeae Strains to more than Five per Cent in Munich

The relative frequency of penicillinase-producing Neisseria gonorrboeae strains isolatedfrom Munich STD patients nowadays clearly exceeds five percent. Penicillin resistance is either due to the 3.2 or 4.4 Megadalton plasmid. Similar trends are reported from other European countries. Therefore, treatment with third generation cephalosporins such as ceftriaxone, cefotaxime or others is now generally advisable not only in the Far East and Central Africa but also in Central Europe.

本邦におけるアフリカ型PPNGの検出

The Plasmid DNA patterns of 22 penicillinase-producing Neisseria gonorrhoeae (PPNG) strains isolated from male patients with gonococcal urethritis in 1988 at Tokyo Metropolitan Taito Hospital were determined. The 3.3 megadalton plasmid, the so called the African type plasmid, was present in 3 isolates out of 22 PPNGs, all of which occurred with a conjugative 24.5 megadalton plasmid. 4.4 megadalton plasmid was present in 19 isolates out of 22 PPNGs, 5 of which occurred with a conjugative 24.5 megadalton plasmid. The infective sources of the 3 cases of the African type PPNGs were found as 1 from philippines and 2 from prostitutes, so called Soap Land Girl in Japan. And they suggests that the African type PPNGs are already common in Japan.

Intrinsic Penicillin Resistance in Penicillinase‐Producing Neisseria gonorrhoeae Strains

The minimal inhibitory concentrations (MICs) of ampicillin for fifty strains of beta-lactamase-producing Neisseria gonorrhoeae (PPNG) isolated in Japan ranged from 1.56 to 200 micrograms/ml, and all the strains harbored a 4.5 megadalton plasmid. These strains were classified into two groups: dicloxacillin-susceptible (28%) and -resistant group (72%). A linear correlation was found in the dicloxacillin-susceptible strains between their beta-lactamase activity and the susceptibility to ampicillin, but not in the dicloxacillin-resistant strains. This suggests that the high ampicillin resistance in PPNG is due not only to acquiring the beta-lactamase producing plasmid, but also to some intrinsic resistance of the strains. To investigate a cause of the high ampicillin resistance, the beta-lactamase-producing plasmid, pTMS1, was transferred by conjugation to a penicillin-susceptible gonococcal strain as well as to its isogenic multiply antibiotic-resistant transformants, and the susceptibility of the transconjugants to ampicillin was determined. Acquisition of pTMS1 by a penicillin-susceptible strain resulted in a 32-fold increase in resistance to ampicillin, whereas the increase was 128-fold for its isogenic strains which contain some chromosomal mutations. These results suggest that reduced permeability of the outer membrane to ampicillin underlies the high ampicillin resistance of PPNG.

A unique plasmid profile characterizing Salmonella enteritidis isolates from patients and employees in a hospital

Plasmid profiling was used as an epidemiological tool during a period of frequent Salmonella enteritidis infection in a hospital. S. enteritidis was isolated from 22 patients and employees. Isolates from 18 persons harbored one 29 and one 36 megadalton (MDa) plasmid. The 29 MDa plasmid has not been previously described in this species and was not found in 54 control strains of S. enteritidis from other sources. The respective restriction endonuclease digest fragments of the 36 and the 29 MDa plasmids were always identical. This plasmid pattern thus served as a marker for the isolates from the outbreak.

Plasmids in Actinobacillus actinomycetemcomitans strains isolated from periodontal lesions of patients with rapidly destructive periodontitis.

Several strains of Actinobacillus actinomycetemcomitans newly isolated from periodontal lesions of patients with rapidly destructive periodontitis were all shown to possess identical plasmid profiles consisting of 4 plasmids. The largest plasmid, 20 MegaDalton (MDa), was also found in reference strains. Two different methods were used for isolation of the plasmids; the large 20 MDa plasmid (pHRP1) was found using the Kado and Liu method only. The 3 small plasmids of 7.0, 5.2 and 4.0 MDa (pHRP2, pHRP3, pHRP4), respectively, were seen using the Birnboim and Doly method. These plasmids are so far to be regarded as cryptic; no phenotypical characters have been linked to their presence. The large 20 MDa plasmid was found in all strains examined, and may be a genotypical marker for the A. actinomycetemcomitans species.

Occurrence of heterogeneous R-plasmids during two concurrent epidemics due to multidrug-resistantSalmonella oranienburg

During two different epidemics that started in August–September, 1980, 140 and 6 multidrug-resistant strains ofSalmonella oranienburg were isolated from a Children's Hospital in New Delhi (epidemic 1) and H.N. Das Hospital in Bombay (epidemic 2) respectively. Out of 140, 123 strains showed high levels of resistance to ampicillin, kanamycin, streptomycin, sulfamethoxazole, tetracycline, and trimethoprim. All six strains of epidemic 2 and two strains of epidemic 1 were also found resistant to chloramphenicol in addition to the above antibiotics. The genetic characterization of conjugative R-plasmids harbored by these strains revealed that, while strains of epidemic 1 carry a fi−, 96 megadalton, an unclassified plasmid, the strains isolated during epidemic 2 contain a fi−, 62 megadalton plasmid of incompatibility group I1. All the six strains of epidemic 2 were found to produce bacteriocin of Col Ib group. Plasmid transfer studies revealed that the genes for antibiotic resistance and bacteriocin production were coded on a single plasmid of 62 megadalton. The data show the significance of detailed genetic analysis while dealing with the clonal outbreaks due to same resistant serotypes.

Characterisation by plasmid profiles, serogroups, and auxotypes of Neisseria gonorrhoeae from Harare, Zimbabwe.

(ABSTRACTOne hundred and fifty five strains of Neisseria gonorrhoeae were regrown from 216 freeze dried cultures originally isolated in Zimbabwe. The gonococci were from men (61 strains) and women (39 strains) attending a referral sexually transmitted diseases (STD) clinic, from women presenting for delivery at hospital with signs of sepsis (22 strains) or with an asymptomatic infection (16 strains), and from babies with ophthalmia neonatorum (17 strains). Seventy five of the 100 isolates from STD clinic patients and 29 of the 55 isolates from hospital patients were penicillinase producing N gonorrhoeae (PPNG). Two thirds of all PPNG strains contained the 24.5 megadalton conjugative plasmid. The 3.2 megadalton resistance plasmid, usually associated with PPNG strains originating in Africa, was present in only one third of the PPNG strains. The 2.6 megadalton cryptic plasmid was present in all strains. Prototrophic and proline requiring auxotypes predominated in both PPNG and non-PPNG strains. Arginine requiring auxotypes were found in four of the 16 isolates from asymptomatic women, whereas three of the 22 strains from women with puerperal sepsis and four of the 61 strains from men with urethritis required both proline and arginine. Fifty eight out of 66 PPNG strains with the 4.4 megadalton plasmid required proline compared with 22/38 PPNG strains with the 3.2 megadalton plasmid and 20 of the 51 non-PPNG strains. Three quarters (38/51) of non-PPNG strains belonged to serogroup WII/III as did 42/66 PPNG strains with the 4.4 megadalton plasmid but only 10/38 PPNG strains with the 3.2 megadalton plasmid. In all, 23 different strain types could be recognized on the basis of plasmid content, auxotype, and serogroup. There was, however, a high degree of homogeneity between PPNG and non-PPNG isolates.

14 ADHESION OF YERSINIA ENTEROCOLITICA TO HELA CELLS AND RABBIT SMALL INTESTINE

The presence of a 40-50 megadalton plasmid is a prerequisite for the expression of virulence of Yersinia enterocolitica. Both chromosomal and plasmid genes seem to be involved in controling the interaction between host cells and the bacterium. In this study, the adhesive properties of Y. enterocolitica were studied by means of two different in vitro assay systems. Adhesion to human epithelial cells was measured by direct microscopy of HeLa cells cultivated on coverslips and incubated with bacteria at 37°C. The capability to adhere to the small intestine of rabbits was evaluated by incubation of small disks of intestine with 3M-labeled bacteria at 37°C, followed by washing and finally determination of the retained, intestinal-associated radioactivity by means of liquid scintilation. The bacterial strains studied were Y. enterocolitica serotype 0:3, harbouring the virulence plasmid (Ye03P+), its isogenic plasmidless derivate (Ye03P-), and the non-pathogenic plasmidless serotype 0:7 (Ye07P-). Ye03P+ as well as Ye03P- adhered extensively to HeLa cells; Ye07P- did not adhere at all. However, while adherence of Ye03P+ to disks of intestinal tissue was considerable, Ye03P- and Ye07P- both adhered to only a small degree. Thus, this study demonstrates that even if pathogenic strains of Y. enterocolitica are capable of adhering to HeLa cells by means of chromosomal genes alone, plasmid encoded properties are necessary for adhesion to the intact mucosal surface, indicating that the binding to cell culture lines differ from the binding to intestine.

Characterisation of penicillinase producing gonococci isolated in Munich, 1981-6.

Twenty three penicillinase producing strains of Neisseria gonorrhoeae (PPNG) isolated in Munich from 1981 to 1986 were characterised in terms of their plasmid content, auxotypes, protein I serovars, and sensitivity to antibiotics. Sixteen strains (70%) harboured a 4.4 megadalton plasmid, with 13 of these strains also containing a 24.4 megadalton plasmid. Seven strains (30%) contained a 3.2 megadalton plasmid, with only one of them containing a 24.4 megadalton plasmid. Thirteen strains (57%) were prototrophic, eight (35%) required proline, one required arginine, and one required proline and arginine for growth. Serovar analysis showed that 16 strains (70%) belonged to the protein IB serogroup and comprised four serovars (IB-1, IB-5, IB-7, and IB-8). The seven strains belonging to the IA serogroup also comprised four serovars (IA-2, IA-4, IA-6, and IA-10). Although 18 different categories of plasmid, auxotype, and serovar were found in the 23 PPNG strains, there was a similarity between the auxotypes in terms of the range of serovars they comprised. Measurement of minimum inhibitory concentrations (MICs) of antibiotics for the strains showed that they were all susceptible in vitro to spectinomycin (MIC less than or equal to 32 mg/l), enoxacin (MIC less than or equal to 0.25 mg/l), cefotaxime, ciprofloxacin, and ceftriaxone (MICs less than or equal to 0.064 mg/l), whereas about half showed some resistance to tetracycline or cefotiam, or both.