Characterization of R-plasmids in environmental isolates ofsalmonella: Host range and stability

Four environmental isolates ofSalmonella, resistant to several drugs, were examined for plasmid carriage with four different plasmid DNA isolation procedures. The method of Birnboim and Doly gave the best results. Three of the strains possessed a single plasmid with molecular weights of 60 (kanamycin resistant), 44.5 (kanamcin resistant), and 23.4 Md (ampicillin and amoxicillin resistant); the other strain (resistant to tetracycline) harbored two plasmids of 69.8 and 2.2 Md. The 69.8 Md was the one responsible for resistance. All plasmids were fi^–, and the 44.5 Md Kc^r plasmid synthesized a sex pilus type F. Some properties related to the dissemination of R-plasmids, such as host range, transfer frequencies, and in vitro stability, were studied. Plasmids generally showed a wide host range and high stability in the transconjugants tested. It could be concluded that these plasmids may be widely disseminated in the environment studied.     

Transfer of the drug-resistance transposon Tn5 toErwinia herbicola and the induction of insertion mutations

Translocatable drug-resistance element (transposon) Tn5 was transferred through conjugation from its carrier suicidal plasmid pJB4JI, harbored byEscherichia coli, toErwinia herbicola. The frequencies of transfer ranged from 0.4×10^–8 to 26×10^–8 per recipient cell. Membrane filter mating yielded more transconjugants than the spread plate technique. Several insertion mutations resulting in loss of bacteriocinogenicity were detected. The location of Tn5 in the mutant genomes was determined by Southern blot hybridization using Tn5-containing pRZ102 as the³²P-labelled probe. The resulting autoradiogram showed specific hybridization with a 96 megadalton plasmid in nine mutants ofErwinia herbicola out of ten tested. However, in one mutant, the 96 megadalton plasmid was missing; instead a larger plasmid containing Tn5 was apparent which was not present in the original strain. This plasmid may have arisen by dimerization of the 96 megadalton plasmid as a result of Tn5 insertion. Using this method, we show that the insertion of Tn5 in that plasmid may have caused the loss of bacteriocinogenicity. The potential usefulness of this technique in genetic analyses of otherErwinia species and other phytobacteria is discussed.     

Double Infection,Recombination, and Segregation of Two R Factors,R 100 and Rts1, and Their Possible Bearing on the Genetic Structure of R 100

Applying the observation by Yokota et al (1969) that a cell doubly harboring an R factor (R100) and a temperature sensitive R factor (Rts1) produces segregant R factors with various resistance patterns, a total of 271 segregant R factors were obtained. There were 163 resistant to (sul, str, kan), 39 resistant to (sul, str, cml, kan), 62 resistant to (sul, str, tet, kan) and finally 7 resistant to (tet, kan). More than 90% of the former 3 segregants were fi⁺ and the remainder, including all of the (tet, kan) segregants, were fi^?. Some fi^? segregants with the former 3 resistance patterns and all of the (tet, kan) segregants were nontransmissible. All of these segregants were still temperature sensitive. Based upon the results of three experiments; (a) the growth at 43 C to observe linked loss of the kan gene and the genes derived from R 100, (b) a conjugal analysis of the relevant resistant markers, and (c) a transductional analysis of these same markers, several conclusions were made. The 2 R factors both consisting of a circle were supposed to have recombined to form a larger circle which then further resulted in the final formation of smaller circles. The possible bearing of these observations and conclusions on the genetic structure of R 100 was discussed.     

Molecular studies of FIme resistance plasmids, particularly in epidemic Salmonella typhimurium.

Summary The molecular sizes of F₁ me resistance plasmids from strains of Salmonella typhimurium, S. wien and S. typhi were within the range 87.9–102.6×10⁶ daltons. DNA reassociation studies indicated that the plasmids from these hosts had at least 80% of their nucleotide sequences in common. A high proportion of F₁ me plasmids cannot mediate their own transfer. The non-autotransferring property of such plasmids is the result of DNA deletion; a non-autotransferring F₁ me plasmid was about 10×10⁶ daltons shorter than autotransferring representatives of the group, and its DNA showed 100% homology with them. Plasmids of the F₁ me group are incompatible with the F factor and with F₁R factors. F₁ me plasmids are incompatible with the fi ⁺ MP10 plasmid of S. typhimurium, whereas F and F₁ factors are compatible with MP10 (Anderson et al., 1977). There was no significant DNA homology between members of the F₁ me group and MP10, and these plasmids may share only a small region of DNA responsible for their incompatibility. The F₁ me R factors examined had 29–37% DNA homology with the F factor, and 50–58% homology with the F₁ resistance plasmid, R162. Molecular examination therefore supports the genetic differentiation of members of the F₁ me group from other F-like plasmids. Both types of investigation can thus be used in epidemiological studies of bacterial strains carrying resistance or other plasmids.     

Application and evaluation of the phage resistance- and bacteriocin-encoding plasmid pMRC01 for the improvement of dairy starter cultures.

The conjugative 63-kb lactococcal plasmid pMRC01 encodes bacteriophage resistance and production of and immunity to a novel broad-spectrum bacteriocin, designated lacticin 3147 (M.P. Ryan, M.C. Rea, C. Hill, and R.P. Ross, Appl. Environ. Microbiol. 62:612-619, 1996). The phage resistance is an abortive infection mechanism which targets the phage-lytic cycle at a point after phage DNA replication. By using the genetic determinants for bacteriocin immunity encoded on the plasmid as a selectable marker, pMRC01 was transferred into a variety of lactococcal starter cultures to improve their phage resistance properties. Selection of resulting transconjugants was performed directly on solid media containing the bacteriocin. Since the starters exhibited no spontaneous resistance to the bacteriocin as a selective agent, this allowed the assessment of the transfer of the naturally occurring plasmid into a range of dairy starter cultures. Results demonstrate that efficient transfer of the plasmid was dependent on the particular recipient strain chosen, and while high-frequency transfer (10(-3) per donor) of the entire plasmid to some strains was observed, the plasmid could not be conjugated into a number of starters. In this study, transconjugants for a number of lactococcal starter cultures which are phage resistant and bacteriocin producing have been generated. This bacteriocin-producing phenotype allows for control of nonstarter flora in food fermentations, and the phage resistance property protects the starter cultures in industry. The 63-kb plasmid was also successfully transferred into Lactococcus lactis MG1614 cells via electroporation.     

R-Plasmid Transfer to and from Escherichia coli Strains Isolated from Human Fecal Samples

Strains of Escherichia coli recently isolated from human feces were examined for the frequency with which they accept an R factor (R1) from a derepressed fi⁺ strain of E. coli K-12 and transfer it to fecal and laboratory strains. Colicins produced by some of the isolates rapidly killed the other half of the mating pair; therefore, conjugation was conducted by a membrane filtration procedure whereby this effect was minimized. The majority of fecal E. coli isolates accepted the R factor at lower frequencies than K-12 F⁻, varying from 10⁻² per donor cell to undetectable levels. The frequencies with which certain fecal recipients received the R-plasmid were increased when its R⁺ transconjugant was either cured of the R1-plasmid and remated with the fi⁺ strain or backcrossed into the parental strain. The former suggests the loss of an incompatibility plasmid, and the latter suggests the modification of the R1-plasmid deoxyribonucleic acid (DNA). In general, the fecal R⁺E. coli transconjugants were less effective donors for K-12 F⁻ and heterologous fecal strains than was the fi⁺ K-12 strain, whereas the single strain of Citrobacter freundii examined was generally more competent. Passage of the R1-plasmid to strains of salmonellae reached mating frequencies of 10⁻¹ per donor cell when the recipient was a Salmonella typhi previously cured of its resident R-plasmid. However, two recently isolated strains of Salmonella accepted the R1-plasmid from E. coli K-12 R⁺ or the R⁺E. coli transconjugants at frequencies of 5 × 10⁻⁷ or less.     

Plasmid profiles ofLegionella species

Forty-six strains ofLegionella species were assayed for plasmid DNA content using routine laboratory procedures. Large-molecular-weight cryptic plasmids were detected inLegionella pneumophila serogroups 2, 3, and 4,L. bozemanii, L. dumoffii, L. micdadei, L. gormanii, L. longbeachae, and as yet unclassifiedLegionella-like organisms. No plasmids were found in strains ofL. pneumophila serogroups 1, 5, and 6. No correlations could be made between the possession of a specific plasmid profile, or lack of one, and any phenotypic markers such as virulence or antibiotic resistance. Several parameters were identified in this study as critical to the isolation of plasmid DNA fromLegionella: (i) DNA preparations obtained from frozen egg or animal materials had a higher incidence of detectable plasmid DNA than subcultures on bacteriologic media. (ii) A newly formulated broth supported exponential growth in all of the 46 strains; one strain required the addition of CO₂. (iii) Considerable heterogeneity was seen in cell susceptibility to various detergents. Since no single lytic agent was suitable for all strains, both ionic and noninic lysis methods were used with each strain. Within the limitations of both crude lysate preparations and the agarose gel electrophoresis method, this study identified a large 60–80 megadalton plasmid species in over 50% of the plasmid-containing strains.     

Plasmid detection in a bacteriocinogenic strain of Clostridium perfringens

Bacteriocinogenic Clostridium perfringens, strain 28, harboured plasmid DNA detectable by dye-bouyant density-gradient centrifugation. This plasmid DNA was absent from an ultraviolet light cured variant which had simultaneously lost its immunity and ability to produce bacteriocin. Agarose gel electrophoresis of the plasmid DNA revealed at least six bands but denaturation experiments suggested three plasmids occurring in more than one conformation. Electron microscopy revealed three major size distributions of circular DNA of molecular weights 1,5,6, and 7.1 megadaltons. Some evidence suggests that the 5.6 megadalton plasmid may control bacteriocin 28 production.     

Isolation and molecular characterization of vancomycin-resistant Enterococcus faecium in Malaysia

Nineteen strains of vancomycin-resistant Enterococcus faecium isolated from 10 of 75 (13.3%) tenderloin beef samples were examined for resistance to selected antibiotics, presence of plasmids, and genetic diversity by random amplification of polymorphic DNA analysis. All strains showed multiple resistant to the antibiotics tested. Multiple antibiotic indexing of the vancomycin-resistant E. faecium strains showed that all (100%) originated from high risk contamination environments where antibiotics were often used. Plasmids ranging in size from 1.5 to 36 megadalton were detected in 15 of 19 (79%) strains. Thus, three plasmid profiles and eight antibiotypes were observed among the E. faecium strains. A high degree of polymorphism was obtained by combining the results of the two primers used; with the 19 E. faecium strains being differentiated into 19 RAPD-types. These preliminary results suggest that RAPD-PCR has application for epidemiologic studies and that resistance patterns and plasmid profiling could be used as an adjunct to RAPD for the typing of E. faecium in the study area.     

Selection and properties of spontaneous mutants ofListeria monocytogenes ATCC 15313 resistant to different bacteriocins produced by lactic acid bacteria strains

The frequency of spontaneous mutants ofListeria monocytogenes ATTC 15313 resistant to the inhibitory action of three bacteriocins of lactic acid bacteria previously discovered in our laboratory (mesenterocin 52, curvaticin 13, and plantaricin C19) was estimated to be in the range of 10^–3 to 10^–4. The phenotypic character of resistance was stable during several generations in the absence of contact with bacteriocins. The resistance was not due to the inactivation of bacteriocins nor to a modification of their adsorption on the target cells. The selected mutants resistant to one of the bacteriocin cited above showed a cross-resistance to the two other bacteriocins, but not to nisin.     

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 (Lap^r), sucrosefermenting (Suc⁺) wild strain, its derivatives, and pGB 301 erythromycin resistance plasmid (Ery^r) at the frequencies of 10⁴ transformants/g 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^–Ery^r, Lac⁺Suc⁺Lap⁺Lap^rEry^r, and Lac^–Suc⁺Lap⁺ Lap^rLap^r, confirmed that 2.0 and 65.0 megadalton (MDa) plasmids carried genes for Suc⁺Lap⁺Lap^r 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.     

Exploitation of Plasmid pMRC01 To Direct Transfer of Mobilizable Plasmids into Commercial Lactococcal Starter Strains

Genetic analysis of the 60.2-kb lactococcal plasmid pMRC01 revealed a 19.6-kb region which includes putative genes for conjugal transfer of the plasmid and a sequence resembling an origin of transfer (oriT). This oriT-like sequence was amplified and cloned on a 312-bp segment into pCI372, allowing the resultant plasmid, pRH001, to be mobilized at a frequency of 3.4 × 10⁻⁴ transconjugants/donor cell from an MG1363 (recA mutant) host containing pMRC01. All of the resultant chloramphenicol-resistant transconjugants contained both pRH001 and genetic determinants responsible for bacteriocin production and immunity of pMRC01. This result is expected, given that transconjugants lacking the lacticin 3147 immunity determinants (on pMRC01) would be killed by bacteriocin produced by the donor cells. Indeed, incorporation of proteinase K in the mating mixture resulted in the isolation of transformants, of which 47% were bacteriocin deficient. Using such an approach, the oriT-containing fragment was exploited to mobilize pRH001 alone to a number of lactococcal hosts. These results demonstrate that oriT of pMRC01 has the potential to be used in the development of mobilizable food-grade vectors for the genetic enhancement of lactococcal starter strains, some of which may be difficult to transform.     

Molecular studies of an fi+ plasmid from strains of Salmonella typhimurium

Summary Plasmid DNA has been isolated from five fi ⁺ strains of Salmonella typhimurium of independent origin, including type 36 and LT2. The mean contour length of the plasmids was between 27.3 and 29.3 m. A variant line of S. typhimurium type 36 which was fi ^- yeilded no plasmid DNA. These results support the hypothesis that the fi ⁺ property of S. typhimurium is coded by a plasmid. In S. typhimurium 36 this plasmid, designated MP10₃₆, also appears to code for restriction of non-donor-specific phages. Molecular studies indicate that superinfection of S. typhimurium 36 with the kanamycin resistance determinant K, which results in loss of the fi ⁺ property, is correlated with loss of MP10₃₆. Reassociation experiments demonstrate a high degree of homology between the DNA of all five S. typhimurium plasmids, and between MP10₃₆ and K. MP10₃₆ has some homology with F and F-like R factors, but not with plasmids of other compatibility groups. A recombinant between an ampicillin resistance determinant and MP10₃₆ is autotransferable at low frequency. The significance of these findings is discussed.     

Plasmid mediated metal and antibiotic resistance in marinePseudomonas

Pseudomonas sp isolated from the Bay of Bengal (Madras coast) contained a single large plasmid (pMR1) of 146 kb. Plasmid curing was not successful with mitomycin C, sodium dodecyl sulfate, acridine orange, nalidixic acid or heat. Transfer of mercury resistance from marinePseudomonas toEscherichia coli occurred during mixed culture incubation in liquid broth at 10^–4 to 10^–5 ml^–1. However, transconjugants lacked the plasmid pMR1 and lost their ability to resist mercury. Transformation of pMR1 intoE. coli competent cells was successful; however, the efficiency of transformation (1.49×10² Hg^r transformants g^–1 pMR1 DNA) was low.E. coli transformants containing the plasmid pMR1 conferred inducible resistance to mercury, arsenic and cadmium compounds similar to the parental strain, but with increased expression. The mercury resistant transformants exhibited mercury volatilization activity. A correlation existed between metal and antibiotic resistance in the plasmid pMR1.     

Analysis of pristinamycin-resistant Staphylococcus epidermidis isolates in the Tunisian Bone Marrow Transplant Center

Aims: We report the analysis of genetic determinants conferring resistance to pristinamycin in Staphylococcus epidermidis strains and epidemiology typing of these strains by pulsed‐field gel electrophoresis. Methods and Results: Staphylococcus epidermidis (346 isolates) were searched for strains with pristinamycin resistance. Pristinamycin‐resistant strains (seven isolates) were isolated in five patients with haematological cancer in the Bone Marrow Transplant Centre of Tunisia in 2002. Resistance to pristinamycin was observed in 2% of isolates. The seven pristinamycin‐resistant strains shared resistance to oxacillin (MIC = 8–512 μg ml^?1), gentamicin (MIC = 16–512 μg ml^?1), erythromycin (MIC > 1024 μg ml^?1), lincomycin (MIC > 1024 μg ml^?1), pristinamycin (MIC = 4–16 μg ml^?1) and rifampin (MIC = 128–256 μg ml^?1). erm genes were amplified: ermA from six strains and ermC from one. vga gene encoding streptogramins A resistance (pristinamycin résistance) was amplified from all strains and typed as vgaA by analysis after electrophoresis of restriction profiles of vga amplicons (two fragments with Sau3A of 164 and 378 bp; one fragment with EcoRI). Pulsed‐field gel electrophoresis (PFGE) of SmaI chromosomal DNA digests of the seven S. epidermidis isolates divided them into two distinct pattern types: pulsed‐field type A (classified from A1 to A6 subtypes) and type B. The six strains harbouring ermA genes belonged to the PFGE type A while the strain harbouring ermC genes belonged to the PFGE type B. We characterized an epidemic strain carrying the vgaA and ermA genes responsible for the outbreak. Conclusions: Two clones of pristinamycin‐resistant S. epidermidis were isolated in our patients. One of them, isolated in all patients, had expanded over six months suggesting acquisition by cross‐contamination. Significance and Impact of the study: Increasing isolation of pristinamycin resistant S. epidermidis strains is an alarming indicator of nosocomial dissemination. The vector will be determined to establish a system of epidemiological surveillance.     

Characterization of Recurrent and Sporadic Listeria monocytogenes Isolates from Raw Milk and Nondairy Foods by Pulsed-Field Gel Electrophoresis, Monocin Typing, Plasmid Profiling, and Cadmium and Antibiotic Resistance Determination

Following previous surveys to assess the incidence of Listeria monocytogenes in raw milk and nondairy foods processed in Northern Ireland, isolates were characterized as recurrent or sporadic on the basis of multilocus enzyme electrophoresis (MEE) analysis and restriction fragment length polymorphism typing. In the present study, 45 representative recurrent and sporadic electrophoretic types (ETs) previously identified by MEE were subjected to pulsed-field gel electrophoresis (PFGE) of genomic DNA macrorestriction fragments, monocin typing, plasmid profiling, and an examination of resistance to cadmium and nine different antibiotics. Although PFGE proved to be capable of subdividing a number of recurrent and sporadic ETs, the grouping of strains arrived at by PFGE and MEE were in broad agreement, and previous conclusions regarding the designation of L. monocytogenes strains as recurrent or sporadic remained unaltered. It is considered that PFGE was able to detect minor genetic changes in recurrent ETs which occurred during the time period in which food surveys were carried out. Production of type E monocin (Types A to E were found among the 45 strains), plasmid carriage, and resistance to cadmium occurred more frequently in recurrent than in sporadic strains and may be important with regard to the ability of L. monocytogenes to persist in food and food-processing environments. Only 2 of 45 strains showed resistance to any of the nine antibiotics tested: two sporadic strains were resistant to tetracycline (MIC, 64 μg ml⁻¹).     

Bacteriocin plasmids ofPediococcus acidilactici

Summary Pediococcus acidilactici strains E, F and H isolated from fermented sausages produced bacteriocins which were protein in nature and inhibitory to a variety of spoilage and pathogenic microorganisms often encountered in foods. These strains harbored two to three plasmids ranging in size from 7.4 to 40.2 megadaltons. Curing experiments and plasmid profile analysis indicated the involvement of plasmid DNA with bacteriocin activity in all three strains. Carbohydrate fermentation and antibiotic resistance phenotypes did not appear to be associated with bacteriocin plasmids. Both bacteriocin activity and resistance determinants were linked in strain H and mediated by a 7.4-megadalton plasmid, whereas in strains E and F these two traits were not linked.     

Organophosphorus and carbamate resistance in the predacious miteTyphlodromus pyri due to insensitive acetylcholinesterase

The cause of parathion and propoxur resistance inTyphlodromus pyri was studied in a Dutch strain in which resistance was dependent on a semi-dominant gene. Activity of glutathione S-transferase and acetylcholinesterase and reaction rate of acetylcholinesterase with paraoxon and propoxur were measured in this resistant (R) and in a susceptible (S) strain. The R strain was 100-fold resistant to parathion and 2300-fold resistant to propoxur. A 36-fold reduction was found in rate of inhibition of acetylcholinesterase in the R strain for paraoxon, and a 14-fold reduction for propoxur. In combination with the monogenic nature of the resistance, this proves that the insensitivity of acetylcholinesterase is the cause of resistance. The rate constant of acetylcholinesterase inhibition at 25°C in the S and R strains was 1.5×10⁵ and 4.2×10³ M ^–1 min^–1 respectively for paraoxon, and 5.1×10⁴ and 3.6×10³ M ^–1 min^–1 for propoxur. There was no significant difference between the R and S strains in glutathione S-transferase activity. The R strain had a somewhat lower acetylcholinesterase activity than the S strain.     

Influence of pH on growth and bacteriocin production byLactococcus lactis subsp.lactis 14ONWC during batch fermentation

The influence of pH on growth, and lactic acid and bacteriocin production byLactococcus lactis subsp.lactis 140 NWC was studied during batch fermentation in a lactose-based complex medium. Growth and lactic acid production were modelled using a simple logistic equation while substrate consumption was found to be a function growth and lactic acid production rate. The optimal pH for growth and lactic acid production was between 6.0 and 6.5. Bacteriocin production showed primary metabolite kinetics. pH had a dramatic effect on the production of the bacteriocin, lactococcin 140. A maximum activity of 15.4 × 10⁶ AU (arbitrary units) 1^–1 was obtained after 7 h at pH 5.5. Maximum bacteriocin activity was achieved before the end of growth and was followed by a decrease in activity, which was due to adsorption to the cells of the producing organism, possibly followed by degradation by specific proteases. Both bacteriocin production and degradation rates were higher at pH 5.0 and 5.5, resulting in sharper activity peaks than at pH 6.0 or 6.5. On the basis of the experimental results a qualitative model for bacteriocin production is proposed.