Characteristics of penicillinase plasmids from Staphylococcus aureus strains

Penicillinase plasmids are present in most MRSA strains. They are very varying in their genotype and phenotype they confer. Penicillinase plasmids were transduced from 80 hospital MRSA strains to NCTC 8325 and the phenotype as well as the incompatibility group of plasmid were determined. Resistance to cadmium (high and low level), resistance to organic and nonorganic mercury compounds, arsenate/arsenite/antimonium resistance, resistance to bismuth and hypersensitivity to bismuth, resistance to macrolides as well as beta-lactamase production and its inductibility were checked. Among the examined strains 20 different phenotypes of penicillinase plasmids were found. Patterns of penicillinase plasmids were compared to DNA patterns of the investigated strains after digestion with SmaI and separation in pulsed field electrophoresis (PFGE). It was shown that strains with the same PFGE pattern often differ in the type of their penicillinase plasmid. Determining of penicillinase plasmid phenotype could be useful in differentiating S. aureus strains sharing the same pattern of PFGE.     

Studies on plasmid replication. III. Isolation and characterization of replication-defective mutants

Some 85 Staphylococcus aureus mutants phenotypically thermosensitive for penicillinase plasmid segregation (Seg-) have been isolated and characterized. Some of the mutations were plasmid-linked and those studied in detail were found to be defective in plasmid replication, most problbly at the initiation stage. Analysis of the segregation behavior of these mutants suggested a figure of 2.7 for the average number of plasmid copies per cell in a random culture. Other mutations were host chromosome-linked and these could be divided into at least three classes on the basis of their ability to maintain plasmids of the two different incompatibility sets: some were defective for type I plasmids, some for type II, and some for both types. One host mutant, defective in segregation of type I but not type II plasmids, was defective in polymerization of both.     

Plasmid-linked Resistance to Inorganic Salts in Staphylococcus aureus

The penicillinase plasmids, a series of extrachromosomal resistance factors in Staphylococcus aureus, were found to carry determinants of resistance to a series of inorganic ions as well as resistance to penicillin and, in some cases, erythromycin. Most of the ions involved were inhibitory but not lethal to the bacteria; the resistance markers conferred an increase in resistance by comparison with susceptible organisms of between 3- and 100-fold, depending on the ion involved. Separate genetic loci for resistance to arsenate, arsenite, lead, cadmium, mercuric, and bismuth ions were demonstrated. Resistance to antimony and resistance to zinc were also found but were not separated genetically from resistance to arsenite and cadmium, respectively. The ion resistance markers appeared to form a cluster on the plasmid, with no other known marker within it. Naturally occurring plasmids were observed that lacked one or more of these ion resistance markers, as well as penicillinase-negative strains that were resistant to one or more of the ions. The patterns of markers carried by these various strains may provide some understanding of the evolution of a plasmid linkage group.     

Characterization of plasmids that confer inducible resistance to 14-membered macrolides and streptogramin type B antibiotics in Staphylococcus aureus

During a period from 1978 to 1989, 413 Staphylococcus aureus strains were isolated at 27 different geographical regions in Hungary; they exhibited an inducible resistance to the 14-membered macrolides and streptogramin type B antibiotics, but not to the 16-membered macrolides and lincosamides: this resistance is referred to as PMS resistance phenotype. The isolates were mostly associated with patients suffering from staphylococcal diseases and with hygienic screenings in hospitals and closed communities. They were rarely isolated from food-poisoning cases, food hygienic screenings, or animal sources. Strains with PMS resistance phenotype were resistant to penicillin (99.0%), tetracycline (78.7%), and chloramphenicol (63.0%); however, they were susceptible to oxacillin. Most of them (94.2%) belonged to the phage type 52-complex. The determinant for PMS phenotype was located on plasmids, which also encoded beta-lactamase production and cadmium ion resistance, but not arsenate resistance. Three types of plasmid with molecular size of 50 kilobases (kb), 23.8 kb, and 16.8 kb, were found among the strains with PMS resistance phenotype, and the 50 kb and 23.8 kb plasmids also encoded mercury resistance. The 16.8 kb and 23.8 kb plasmids belonged to incompatibility group 1.     

Recombination between plasmids of incompatibility groups P-1 and P-2.

R plasmids of incompatibility group P-2 are readily transmissible between Pseudomonas strains, but not to Escherichia coli or other enterobacteria, whereas those of group P-1 have a broad host range. Pseudomonas aeruginosa donor strains carrying both a P-1 plasmid (RP1, RP4, or R751) and a P-2 plasmid (pMG1, pMG2, pMG5, or RPL11) were mated with E. coli K-12, and selection was imposed for resistance markers on the P-2 plasmids. Transconjugants were obtained at a low frequency, in which P-2 markers were expressed and were serially transmissible in E. coli together with P-1 markers. These plasmids had P-1 incompatibility properties, conferred susceptibility to phages active on P-1 carrying strains, and behaved on sucrose gradient centrifugation as unimolecular species of higher molecular weights than the P-1 parent. Recombinant plasmid formation was independent of a functional Rec gene in both donor and recipient and, with R751, had a preferred site leading to loss of trimethoprim resistance. Interaction between insertion sequences may be involved. Thus, plasmids of group P-2 can recombine with R factors of another group quite separate in compatibility properties, host range, and pilus type. Formation of such recombinants provides one pathway by which the genetic diversity of plasmids may have evolved.     

Wild-Type Strain of Staphylococcus aureus Containing Two Genetic Linkage Groups for Penicillinase Production

Staphylococcus aureus strain 55C1, isolated from a patient in 1955, contained two genetic linkage groups for penicillinase formation. One was linked to genes that control resistance to cadmium and mercuric ions; it had properties of a plasmidborne gene. The other was not linked to resistance to these metal ions; it had properties of a chromosomal gene. Penicillinase formation by cells that contained either linkage group was inducible by penicillins. Induced penicillinase in cells that contained both linkage groups equalled the sum of that produced in cells containing each group singly. Exopenicillinase produced by cells containing either gene was serological type A. Constitutive penicillinase formation resulting from regulator gene mutations in either linkage group was repressed to differing extents by a wild-type determinant in the trans position. The genetic structure and the regulation of penicillinase formation in strain 55C1 resembled in general those for penicillinase linkage groups which Asheshov and Dyke described for diploid mutant strains of S. aureus PS 80. There were differences in detail, however.     

New R-plasmids in strains of Serratia marcescens with multiple drug resistance

In 4 S. marcescens polyresistant strains isolated from patients conjugative plasmids transferred to Escherichia coli have been detected. Two of these strains carry each one plasmid which codes resistance to 10 different antibiotics, including aminoglycosides which rarely occur in our country, and belongs to group IncC. The third strain is the host of 2 plasmids. One of them is similar to the above-mentioned 2 plasmids with respect to the incompatibility group and a set of markers, but additionally codes resistance to cephalosporins; the second plasmid has been determined as belonging to group IncM, unstable and capable of rendering the cells highly resistant only to aminoglycosides. And, finally, the fourth strain also carries 2 plasmids: one of them is unstable and belongs, supposedly, to group IncI alpha, and the second plasmid is stable and belongs to group IncM. The plasmid of group IncI alpha differs from all other plasmids of our Serratia by its capacity of rendering the cells highly resistant to chloramphenicol.     

Incompatibility and molecular relationships between small staphylococcal plasmids carrying the same resistance marker

Incompatibility relationships between staphylococcal plasmids carrying the same, single resistance marker were studied by means of appropriate recombinant plasmids. Naturally occurring plasmids encoding streptomycin, tetracycline, or chloramphenicol resistance, respectively, were used in this study, four of each phenotype. The plasmids responsible for tetracycline resistance proved to belong to a single incompatibility set. Similarly, the four streptomycin resistance plasmids fall in the same incompatibility set. On the other hand, plasmids encoding chloramphenicol resistance were divided in four distinct incompatibility sets, three of them being newly defined. Study of the molecular relationships between these plasmids by DNA-DNA hybridization and restriction endonuclease cleavage supported the conclusions from genetic tests that the four Tc^r and the four Sm^r plasmids are essentially identical, whereas the four Cm^r plasmids are diverse.     

Characteristics of new pKMR-plasmids detected in natural strains of Enterobacteriaceae

pKMR-plasmids controlling the antibiotic resistance and adhesive properties were isolated from clinical strains of E. coli O26 and O124, and Sh. sonnei. Two of them, i.e. pKMR 207 and pKMR 208 were conjugative. On conjugation they jointly transferred the features of the antibiotic resistance and capacity for production of the colonization antigen. The studies on transformation of E. coli K 12 802 with the plasmid DNA of E. coli O124 showed that the antibiotic resistance and colonization properties in E. coli O124 were controlled by the nonconjugative plasmid pKMR 209. It was found that plasmids pKMR 207 and pKMR 208 had the fi(-)-phenotype. None of the plasmids allotted the host cells sensitivity to the donor specific phages of the incompatibility groups F, N, P, W, and I. Probably, the plasmids did not belong to these incompatibility groups. When the cells of E. coli K 12 802 were transformed with the plasmid DNA of the wild strain to the hemolytic strain of S. typhimurium with multiple antibiotic resistance, 3 pKMR 210 plasmids with different markers of the antibiotic resistance were detected in the transformants. One of the plasmids controlled both the drug resistance and the capacity for production of hemolysin. The ability of the detected pKMR plasmids to inhibit fertility and relation to the donor specific phages was studied.     

An efficient and reproducible method for transformation of genetically recalcitrant bifidobacteria

The epidemic community-associated methicillin-resistant clone Staphylococcus aureus USA300 is a major source of skin and soft tissue infections and involves strains with a diverse set of resistance genes. In this study, we report efficient transduction of penicillinase and tetracycline resistance plasmids by bacteriophages φ80α and φJB between clinical isolates belonging to the USA300 clone. High transduction frequencies (10(-5) - 10(-6) CFU/PFU) were observed using phages propagated on donor strains as well as prophages induced from donors by ultraviolet light. Quantitative real-time PCR was employed to detect penicillinase plasmids in transducing phage particles and determine the ratio of transducing particles in phage lysates to infectious phage particles (determined as approximately 1 : 1700). Successful transfer of plasmids between strains in USA300 clone proves transduction is an effective mechanism for spreading plasmids within the clone. Such events contribute to its evolution and to emergence of new multiple drug-resistant strains of this successful clone.     

Polyresistant Enterobacteriaceae strains and their plasmids in a hospital. Medical and theoretical aspects

The properties and origin of multiple resistant strains of Enterobacteriaceae found in the intestine and nasopharynx of infants admitted to the hospital for premature infants were studied. The strains of E. coli of different serovars isolated at various periods contained similar conjugative R plasmids with a molecular weight of 80 Md belonging to the O incompatibility group controlling resistance to kanamycin and physically independent small plasmids controlling resistance to ampicillin (7 Md) and streptomycin-sulfanilamides (4 Md). Multiple drug resistance in the strains of K. pneumoniae was controlled by single large (100-120 Md) plasmid cointegrates with 6-8 resistance markers. Such cointegrates consisted of several potentially independent plasmids, sometimes dividing on transformation of plasmid DNA of the recipient strains of E. coli K12. The small plasmids controlling resistance to ampicillin and streptomycin-sulfanilamides similar to the respective plasmids of E. coli were the constant components of the plasmids cointegrates. The multiple drug resistance in the above strains was combined with high capacity for colonization in premature infants. The medical staff and mothers were the sources of bacterial strains with single plasmids controlling definite types of resistance. It is suggested that the multiple resistant strains of Enterobacteriaceae are formed in hospital as a result of accumulation of the plasmids or plasmid markers and selection. One of the conditions for successive acquisition of new plasmid markers by definite bacterial strains was their high capacity for colonization in patients, which provided constant contacts and genetic exchange of such strains with a wide range of immigrant strains during colonization in the newly admitted patients.     

Phenotypic Suppression of Methicillin Resistance in Staphylococcus aureus by Mutant Noninducible Penicillinase Plasmids

Methicillin (intrinsic) resistance of Staphylococcus aureus was suppressed almost completely by regulatory gene (penI₁) mutations of penicillinase plasmids that made penicillinase production strictly noninducible. Methicillin resistance was restored by secondary regulatory gene mutations that altered the noninducible phenotype or by complementation with a compatible plasmid that did not bear the noninducible mutation. No evidence was obtained for genetic linkage between a penicillinase plasmid and the gene for methicillin resistance. We suggest, therefore, that the mutant noninducible repressor acted in trans by binding to a site on the methicillin resistance determinant. This hypothesis would imply an appreciable degree of homology between penicillinase plasmids and methicillin resistance genes.     

Characteristics and distribution of plasmids in a clonally diverse set of methicillin-resistant Staphylococcus aureus strains

The aim of this study was to compare the plasmid contents of methicillin-resistant Staphylococcus aureus (MRSA) strains classified into different clonal clusters (CCs). The isolates were collected from 15 Czech hospitals in 2000-2008. Plasmid DNA was detected in 65 (89%) strains, and 33 of them harbored more than one plasmid type. Altogether 24 different types of plasmids were identified, ranging in size from 1.3 to 55 kb. Restriction endonuclease analysis, plasmid elimination, DNA hybridization, and sequencing were used for their further characterization. It has been found that the conjugative, erythromycin resistance and enterotoxin D encoding plasmids are harbored by strains from different CCs. On the other hand, chloramphenicol and tetracycline resistance plasmids, and most of the penicillinase and cryptic plasmids were only detected in certain CCs. Especially, the pUSA300-like plasmids were found exclusively in the USA300 clone strains. The high diversity in plasmid content detected in the study strains implies that plasmids play a major role in evolution of MRSA clonal lineages.     

Spread and evolution of natural plasmids harboring transposon Tn5

Abstract: The presence of transposon Tn 5 was studied in 730 Enterobacteriaceae strains from clinical and sewage origin. From these strains, twenty-five conjugative plasmids harboring transposon Tn 5 were isolated. These plasmids were compared with pJR67 and pRYC119, the only previously studied plasmids harboring Tn 5 . A phylogenetic tree of the evolution of all different plasmids was proposed. Irrespective of their bacterial host and geographical place of isolation, some of the plasmids were shown to be identical. All of them can be included in only eight different prototypical plasmid species. Twenty-two plasmids (88%) carried an IncI1 incompatibility determinant as judged form DNA hybridization experiments. The presence of some other common resistance genes suggested that these plasmids are descendants of a common ancestor. These IncI1 plasmids could be grouped in six prototypical species. The results presented here suggest that Tn 5 spread in nature may be dependent on the conjugative ability of the IncI plasmids harboring the transposon, rather than on the efficiency of Tn 5 transposition between different replicons.     

Genetic Studies on Plasmid-Linked Cadmium Resistance in Staphylococcus aureus

The genetic basis of cadmium resistance conferred by three penicillinase plasmids, PI(524), PI(258), and PII(147), of Staphylococcus aureus was examined by mutation, recombination, and deletion analysis. Three separate loci were identified: cadA, responsible for high-level resistance; cadB, giving a low-level resistance, nonadditive to cadA; and mad, a locus marginally decreasing the cadmium resistance of plasmid-positive staphylococci. The loci cadA and mad were present on all three plasmids, but cadB was only found on PII(147). Spontaneous deletions of mad involved up to three-fourths of the plasmid genome, which allowed derivation of a partial deletion map of PII(147), a plasmid with a contour length of 10.9 mum, corresponding to a molecular weight of 20.4 x 10(6).     

Extrachromosomal Control of Methicillin Resistance and Toxin Production in Staphylococcus aureus

All of 41 naturally occurring coagulase-positive methicillin-resistant strains of Staphylococcus aureus isolated in various laboratories were resistant to several antibiotics and were lipase-negative. Most strains produced hemolysins, and 38 strains produced enterotoxin B. Acriflavine treatment of four strains resulted in elimination of resistance to methicillin and mercury; in one strain, resistance to cadmium was also lost. Production of enterotoxin B and beta-hemolysin was eliminated in all four strains and penicillinase production was eliminated in one strain. In transduction experiments, methicillin resistance and enterotoxin B production were transferred together at a frequency of 0.2 x 10(-8) to 1.1 x 10(-8) by use of ultraviolet-induced phage lysates from naturally lysogenic methicillin-resistant strains. Cotransductions of resistance to mercury and cadmium, as well as production of penicillinase and beta-hemolysin, were obtained to some extent. The extrachromosomal character of these determinants and their possible genetic association are discussed.     

The complete genome sequences of four new IncN plasmids from wastewater treatment plant effluent provide new insights into IncN plasmid diversity and evolution

The dissemination of antibiotic resistance genes among bacteria often occurs by means of plasmids. Wastewater treatment plants (WWTP) were previously recognized as hot spots for the horizontal transfer of genetic material. One of the plasmid groups that is often associated with drug resistance is the incompatibility group IncN. The aim of this study was to gain insights into the diversity and evolutionary history of IncN plasmids by determining and comparing the complete genome sequences of the four novel multi-drug resistance plasmids pRSB201, pRSB203, pRSB205 and pRSB206 that were exogenously isolated from the final effluent of a municipal WWTP. Their sizes range between 42,875 bp and 56,488 bp and they share a common set of backbone modules that encode plasmid replication initiation, conjugative transfer, and plasmid maintenance and control. All plasmids are transferable at high rates between Escherichia coli strains, but did not show a broad host range. Different genes conferring resistances to ampicillin, streptomycin, spectinomycin, sulfonamides, tetracycline and trimethoprim were identified in accessory modules inserted in these plasmids. Comparative analysis of the four WWTP IncN plasmids and IncN plasmids deposited in the NCBI database enabled the definition of a core set of backbone genes for this group. Moreover, this approach revealed a close phylogenetic relationship between the IncN plasmids isolated from environmental and clinical samples. Phylogenetic analysis also suggests the existence of host-specific IncN plasmid subgroups. In conclusion, IncN plasmids likely contribute to the dissemination of resistance determinants between environmental bacteria and clinical strains. This is of particular importance since multi-drug resistance IncN plasmids have been previously identified in members of the Enterobacteriaceae that cause severe infections in humans.     

A method of plasmid classification by integrative incompatibility

A method of plasmid classification by integrative incompatibility has been developed. The characteristics of this system are as follows: (i) The conventional plasmids usually used as standards for incompatibility grouping were integrated into the host chromosome to increase stability and to minimize recombination with the superinfecting plasmid. Strains were constructed by integrative suppression which was in some cases facilitated by the introduction of Tn5 into the plasmid. (ii) The resulting Hfr strains were made deficient in the rec A function to eliminate homologous recombination between the resident and the superinfecting plasmids. A test plasmid is introduced into these rec A Hfr test strains in the stationary phase of growth. In an incompatible cross, the number of transconjugant colonies was usually less than 10^?2 of that in a compatible cross. Occasionally, an inhibitory mechanism, other than incompatibility was coded by the resident plasmid [e.g., restriction in R124 (inc FIV)]. This complicated the interpretation, but did not invalidate the experiment. The colonies arising in incompatible crosses were shown to carry drug resistance determinants coded by both the resident and superinfecting plasmids. These were presumably the result of rec-independent integration of all or part of the superinfecting plasmid into the host chromosome. Thus the reduced frequency of superinfectant formation in an incompatible cross is usually the consequence of incompatibility between the resident and the superinfecting plasmids. This integrative incompatibility system should be useful for epidemiological studies of R plasmids.     

Epidemiological and structural studies of Staphylococcus aureus R plasmids mediating resistance to tobramycin and streptogramin

We have studied the genetic properties of five S. aureus strains (serotypes 18 and III) isolated in France during a recent outbreak and carrying two new resistance characters (tobramycin and streptogramin). After transfer by transduction of these resistances, recipient cells were found in each case to harbor a single multicopy plasmid carrying the two resistance characters plus resistance to cadmium salts and/or resistance to tetracycline. The five Sg^r-Tm^r plasmids recovered by transduction are related epidemiologically; all five belong to the same incompatibility group and they share from 50 to 100% base sequence homology. Three of the five have similar restriction endonuclease patterns.     

Molecular relationships between trimethoprim R plasmids obtained from human and animal sources

A total of 65 trimethoprim R plasmids (35 obtained from human strains and 30 from animal strains of Escherichia coli ) were examined by the technique of restriction endonuclease fingerprinting. Plasmids belonging to incompatibility groups B, F_II and 1Δ obtained from human and animal sources showed close similarities within each group. Plasmids belonging to incompatibility groups 1α and P were also obtained from both human and animal sources, but there was no conclusive evidence of close relationships within the groups. Restriction endonuclease fingerprinting was found to be useful for obtaining information about the epidemiology of R plasmids. Its main limitation in this study related to broad host range plasmids of the P incompatibility group, some of which contained very few sites susceptible to cleavage by the restriction endonucleases tested.