Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Plasmid-associated transfer of tetracycline resistance in Bacteroides ruminicola.

Tetracycline resistance was transferred at frequencies between 10(-7) and 10(-6) per recipient cell in anaerobic matings between two strains of the strictly anaerobic rumen bacterium Bacteroides ruminicola. The donor strain, 223/M2/7, was a multiple-plasmid-bearing tetracycline-resistant strain from the ovine rumen, and the recipient, F101, was a rifampin-resistant mutant of B14, a bovine strain belonging to B. ruminicola subsp. brevis. Resistance transfer could occur in the presence of DNase, but not in dummy mating mixtures in which filtrate from a donor culture replaced donor cells. Acquisition of tetracycline resistance by the recipient was accompanied by the appearance of a 19.5-kilobase pair plasmid (designated pRRI4) which was homologous with a plasmid of similar size and restriction pattern present in the donor strain. A transconjugant (F115) carrying pRRI4 was also able to act as a donor of tetracycline resistance and plasmid DNA in matings with another recipient. Derivatives of F115 that had spontaneously lost tetracycline resistance lacked detectable plasmid DNA. It is concluded that pRRI4 mediated the transfer of tetracycline resistance. Transfer of resistance was not detectably enhanced by pregrowth of the donor in medium containing tetracycline. Transfer of tetracycline resistance was not detected from 223/M2/7 to a strain, 23 belonging to B. ruminicola subsp. ruminicola.     

Construction and properties of an integrable plasmid for Bacillus subtilis.

A plasmid useful for locating the chromosomal site of cloned DNA fragments that lack intrinsic genetic activity, for insertional mutagenesis, for single-copy complementation, and for dominance studies was constructed. Some plasmids containing Bacillus subtilis DNA were only active in transformation when the tetracycline resistance determinant of the plasmid was inactivated. The results suggest that production of the tetracycline gene product is lethal to B. subtilis.     

Bacteriocin and antibiotic resistance plasmids in Bacillus cereus and Bacillus subtilis.

A number of plasmids have been isolated as covalently closed circular DNAs from strains of Bacillus cereus and B. subtilis. From 12 out of 15 strains of B. cereus, plasmids could be isolated. Most of the B. cereus strains contained two or more plasmids. Their molecular weights ranged from 1.6 X 10(6) to 105 X 10(6). Bacteriocin production could be attributed to a 45 X 10(6)-dalton plasmid (pBC7) from B. cereus DSM 336, and tetracycline resistance to a 2.8 X 10(6) plasmid (pBC16) from B. cereus GP7. Two streptomycin-resistant strains of B. subtilis harbored plasmids of 5.2 X 10(6) and 9 X 10(6), respectively, which were, however, not correlated with the antibiotic resistance. The plasmid carrying resistance to tetracycline, pBC16, which was originally isolated from B. cereus, could be subsequently transformed in B. subtilis, where it is stably maintained.     

The cloning of chromosomal DNA associated with methicillin and other resistances in Staphylococcus aureus

Competitive hybridization was used to detect the deletion of chromosomal DNA accompanying the loss of resistance to methicillin (and concomitantly, to cadmium, mercury and tetracycline) from a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA). The method was also used to screen a partial plasmid library of chromosomal HindIII fragments from the MRSA strain. Eight recombinant plasmid clones were identified as containing DNA included in the deletion. These clones were used as probes to screen a phage library of the total DNA of the same MRSA strain, resulting in the isolation of overlapping recombinant phage clones carrying 24 kb of the deleted DNA. Two of the cloned HindIII fragments were associated closely with methicillin resistance, as shown by probing DNA from an independent methicillin-sensitive/resistant transduced strain pair and from two MRSA strains following growth in the presence of high concentrations of methicillin. The endonuclease map of the cloned DNA indicates the presence of four copies of a direct repeat less than 1 kb in size. The map is also consistent with the presence in the chromosome of sequences for mercury resistance (mer A mer B) and for tetracycline-resistance plasmid pT181.     

A Phage-Mediated Transfer of Chromosomally Integrated Tetracycline Resistance Plasmid in Staphylococcus aureus

The ability of a Staphylococcus aureus isolate WBG7416 to transfer its resistance determinants was studied in conjugation and mixed-culture transfer experiments. It carried plasmid-borne resistance to kanamycin, trimethoprim, chloramphenicol, cadmium, propamidine isethionate, and chromosomal resistance to methicillin, gentamicin, streptomycin, erythromycin, clindamycin, tetracycline, and minocycline. It transferred tetracycline resistance in mixed-culture transfer but not in conjugation experiments. DNA-DNA hybridization of genomic DNA from the tetracycline-resistant transferrants against a labeled tetracycline resistance plasmid, pWBG3, probe revealed the presence of an integrated plasmid in their chromosomes. In contrast, no homology to the probe was detected in the chromosome of a tetracycline-resistant mutant of the recipient strain. The results established a role for a bacteriophage in the transfer of chromosomal tetracycline resistance in WBG7416 besides demonstrating the presence of an integrated tetracycline resistance plasmid in the transferrants. It also offered an insight into the nature of the integrated plasmid.     

The genetics of tetracycline resistance in Staphylococcus aureus.

Eighty-one strains ofStaphylococcus aureus that appeared to be tetracycline resistant on the basis of a preliminary disc-diffusion test were examined fro resistance to tetracycline and to the semi-synthetic tetracycline, minocycline. Minimum inhibitory concentration (m.i.c.) values for both drugs were determined after induction of the strains by growth for 2 h in sub-inhibitory concentrations fo tetracycline. Forty-seven strain (58 percent) had m.i.c. values for minocycline of I2.5 MUg/ml or greater, and were considered to be minocycline resistant. An additional ten strains had m.i.c.r greater, and were considered to be minocycline resistant. An additional ten strains had m.i.c.values for minocycline of 3.I25 to 6-25 MUg/ml and were classified as low-level resistant strain. It appears, therefore, that a faily high proportion fo tetracycline-resistant strains isolated at the present time are resistant of concentrations of minocycline unattainbale in vivo with the recommended dosage forthis antibiotic (Fishk & Tunevall, 1969). Transductioal analysis of the genetic determinantswo types of resistance to high concentrations of tetracycline. Strains in the first categroy (A)were inducibly resistant to tetracycline but sensitive to minocycline; in these strains the resistance determinant was plasmid-borne. Strains in the second categroy (B) were resistant to both tetracycline and minocycline and had low induction ratios for tetracycline resistance; the genetic determinant for resistance in these strains was chromosomal. In addition, certain strains incategroy A were found to carry a chromosomal gene controlling low-level resistance to tetracycline and minocycline. This low-level resistance to tetracycline was masked in the presence of the tetracycline plasmide but could be demonstrated after loss of the plasmid. The results suggest that more than one mechanism of resistance to tetracyclines may exist in staphylococci.     

Tetracycline-inducible transfer of tetracycline resistance in Bacteroides fragilis in the absence of detectable plasmid DNA.

Tetracycline resistance of three Bacteroides fragilis strains was shown to be inducible by subinhibitory concentrations of tetracycline. Tetracycline resistance markers could be transferred to another B. fragilis strain by filter mating. The transferability was inducible by subinhibitory concentrations of tetracycline and did not take place in the absence of tetracycline. The optimum concentration of tetracycline for induction of transfer was about 2 microgram/ml. The transfer was shown to be a conjugation-like process requiring cell-to-cell contact between donor and recipient. Screening of parental donor strains for the presence of plasmid DNA did not demonstrate any detectable plasmids in two of the strains. A 3.0-megadalton plasmid, designated pBY5, was present in the third donor strain. Mobilization of pBY5 by another plasmid (pBF4) showed that pBY5 did not carry the genes responsible for tetracycline resistance. It appears that the genes responsible for resistance to tetracycline as well as those responsible for conjugal transfer may be carried on the chromosome in all three donor strains.     

Versatile cloning vector for Pseudomonas aeruginosa.

A pBR322:RSF1010 composite plasmid, constructed in vitro, was used as a cloning vector in Pseudomonas aeruginosa. This nonamplifiable plasmid, pMW79, has a molecular weight of 8.4 X 10(6) and exists as a multicopy plasmid in both P. aeruginosa and Escherichia coli. In P. aeruginosa strain PAO2003, pMW79 conferred resistance to carbenicillin and tetracycline. Characterization of pMW79 with restriction enzymes revealed that four enzymes (BamHI, SalI, HindIII, and HpaI) cleaved the plasmid at unique restriction sites. Cloning P. aeruginosa chromosomal deoxyribonucleic acid fragments into the BamHI or SalI site of pMW79 inactivated the tetracycline resistance gene. Thus, cells carrying recombinant plasmids could be identified by their carbenicillin resistance, tetracycline sensitivity phenotype. Deoxyribonucleic acid fragments of approximately 0.5 to 7.0 megadaltons were inserted into pMW79, and the recombinant plasmids were stably maintained in a recombination-deficient (recA) P. aeruginosa host.     

Physical structure and genetic expression of the sulfonamide-resistance plasmid pLS80 and its derivatives in Streptococcus pneumoniae and Bacillus subtilis

The 10-kb chromosomal fragment of Streptococcus pneumoniae cloned in pLS80 contains the sul-d allele of the pneumococcal gene for dihydropteroate synthase. As a single copy in the chromosome this allele confers resistance to sulfanilamide at 0.2 mg/ml; in the multicopy plasmid it confers resistance to 2.0 mg/ml. The sul-d mutation was mapped by restriction analysis to a 0.4-kb region. By the mechanism of chromosomal facilitation, in which the chromosome restores information to an entering plasmid fragment, a BamHI fragment missing the sul-d region of pLS80 established the full-sized plasmid, but with the sul-s allele of the recipient chromosome. A spontaneous deletion beginning approximately 1.5 kb to the right of the sul-d mutation prevented gene function, possibly by removing a promoter. This region could be restored by chromosomal facilitation and be demonstrated in the plasmid by selection for sulfonamide resistance. Under selection for a vector marker, tetracycline resistance, only the deleted plasmid was detectable, apparently as a result of plasmid segregation and the advantageous growth rates of cells with smaller plasmids. When such cells were selected for sulfonamide resistance, the deleted region returned to the plasmid, presumably by equilibration between the chromosome and the plasmid pool, to give a low frequency (approximately 10(-3) of cells resistant to sulfanilamide at 2.0 mg/ml. Models for the mechanisms of chromosomal facilitation and equilibration are proposed. Several derivatives of pLS80 could be transferred to Bacillus subtilis, where they conferred resistance to sulfanilamide at 2 mg/ml, thereby demonstrating cross-species expression of the pneumococcal gene.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conjugative chromosomal gene transfer in Bacillus subtilis

Conjugative transfer of 20-kb chromosomal fragment carrying genes encoding tetracycline (tet(r)) and lincomycin (lin(r)) resistance in the soil strain Bacillus subtilis 19 is described. Transfer was preceded by this fragment insertion into the large conjugative pl9cat plasmid producing a hybrid plasmid. Insertion frequency was 10(-4)-10(-5). Then genes tet(r) and lin(r) were transferred to the recipient strains. The transfer of chromosomal genes inserted into the plasmid and plasmid gene cat occurred sequentially and resembled sexduction, which represents chromosomal gene transfer by F'- and R' plasmids during conjugation in Escherichia coli and other gram negative bacteria.     

Mutations in Prophage φ11 That Impair the Transducibility of Their Staphylococcus aureus Lysogens for Methicillin Resistance

Methicillin resistance (mec) is not transduced into Staphylococcus aureus 8325-4, but is transduced into this host after it has been lysogenized with phage phi11 and has acquired the penicillinase plasmid pI524 by a separate transduction (Cohen and Sweeney, 1970, 1973). Strain 8325-4 is competent for transformation of typical plasmid or chromosomal markers and for mec only if it is lysogenic for phi11 or a related prophage (Sj?str?m et al., 1974, 1975). A mutant strain of phi11 that was temperature sensitive (Ts) for vegetative multiplication did not mediate competence for transformation of its 8325-4 lysogen if the lysogen had been grown at a nonpermissive temperature (Sj?str?m and Philipson, 1974). We isolated four Ts mutants of phi11 that did not mediate transducibility of their 8325-4(pI524) lysogens for mec after growth at nonpermissive temperatures (40 to 42 degrees C). Transduction of typical plasmid or chromosomal markers was not affected. These phi11-Ts mutants mediated normal competence of their lysogens for transformation of a tetracycline resistance plasmid. Similarly, phi11-Ts mutants that rendered their lysogens temperature sensitive for transformation did not depress the frequency of transduction of mec. These two types of phi11-Ts mutants fell into two different genetic complementation groups that differed in the physiology of deoxyribonucleic acid synthesis and in the time of expression of the mutations during a single-burst growth cycle at a nonpermissive temperature. A virulent mutant of phi11, which plaqued with 100% efficiency on 8325(phi11), also failed to condition strain 8325-4 for transducibility of mec but retained the ability to confer competence for transformation of a tetracycline resistance plasmid. Different genetic loci and physiological functions are involved in phi11 mutations that affect transducibility of mec and those that affect competence for transformation of markers generally in S. aureus 8325-4.     

Tetracycline resistance genes from Bacillus plasmid pAB124 confer decreased accumulation of the antibiotic in Bacillus subtilis but not in Escherichia coli

Expression of tetracycline resistance by genes originating in the Bacillus plasmid pAB124 was examined in both Bacillus subtilis and Escherichia coli host cells. Expression of resistance in B. subtilis by genes from pAB124 was inducible and associated with decreased accumulation of the antibiotic. A fragment of pAB124 carrying the genes coding for tetracycline resistance was cloned into the E. coli plasmid RSF2124. The cloned fragment conferred a low level of resistance in E. coli, but this was not associated with decreased uptake of tetracycline and was not inducible.     

Identification of a Staphylococcus aureus transposon (Tn4291) that carries the methicillin resistance gene(s).

We isolated a transposon (Tn4291) that carries the resistance gene(s) for methicillin in a secondary insertion site on the penicillinase plasmid pI524. Transposition of Tn4291 into pI524 occurred during the transduction of the tetracycline resistance plasmid pSN1 from a methicillin-resistant donor into a recipient that carried the mec allele in the primary site on the chromosome. Insertion of Tn4291 caused extensive rearrangement of pI524 and resulted in the formation of a 27.9-kilobase-pair plasmid (pIT103) which coded for resistance to methicillin and cadmium, but not penicillin. Although resistance to methicillin and cadmium were always linked, Tn4291 was stably maintained only in the presence of a chromosomal mec allele, while in its absence the plasmid was unstable and transposition to the primary site occurred. Subsequently, a 20.1-kilobase-pair plasmid, pIT203, was formed which retained cadmium resistance and regained the ability to express beta-lactamase activity.     

Isolation and characterization of a plasmid involved with enterotoxin B production in Staphylococcus aureus.

Genetic analysis and molecular characterization of plasmid deoxyribonucleic acid (DNA) was performed in a toxigenic isolate of Staphylococcus aureus strain DU4916. Elimination, transduction, and transformation experiments provided us with a series of derivatives similar except for the presence or absence of genes mediating resistance to penicillin (penr), methicillin (mecr), and tetracycline (tetr) and enterotoxin type B (SEB) production (entB+). The derivatives were examined for the presence of a plasmid species which encodes for SEB production. Two distinct species of covalently closed circular DNA of about 2.8 X 10(6) and 0.75 X 10(6) daltons were identified in an ethidium bromide-cured, penicillinase-negative (pens) isolate, SN109 (mecr tetr emtB+). Further segregation of either methicillin resistance or tetracycline resistance or of both together resulted in the loss of SEB production and the disappearance of both plasmids. Transduction from strain SN109 showed that determinants for tetracycline resistance are carried by the 2.8 X 10(6) dalton plasmid. Transformation with covalently closed circular DNA from strain SN109 yielded mecs tetr entB- transformants harboring the tetracycline resistance plasmid alone and mecr tetr entB+ transformants harboring both the tetracycline resistance and the 0.75 X 10(6)-dalton plasmid. Further segregation of methicillin resistance in transformants was not associated with any change in plasmid DNA. The results indicate that a genetic determinant for SEB production is carried by the 0.75 X 10(6)-dalton plasmid. It is possible, however, that this plasmid cannot be maintained in the host independently from the tetracycline resistance plasmid. Methicillin resistance in the strains examined could not be ascribed to any of the covalently closed circular DNA components resolved in strain DU4916.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.     

Evidence for natural transfer of a tetracycline resistance gene between bacteria from the human colon and bacteria from the bovine rumen.

Previously, we demonstrated conjugal transfer of a specially constructed shuttle vector, pRDB5, from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4. We have now shown that naturally occurring gene transfer elements in Bacteroides species and Prevotella ruminicola can also be transferred between these two genera. A self-transmissible chromosomal element originally found in a clinical isolate of Bacteroides fragilis (Tcr Emr 12256) was transferred from B. uniformis 0061 to P. ruminicola B(1)4 and from P. ruminicola B(1)4 back to B. uniformis or to another human colonic species, Bacteroides thetaiotaomicron. Similarly, a conjugative plasmid (pRRI4) originally found in P. ruminicola 223 was transferred from P. ruminicola B(1)4 to B. uniformis or B. thetaiotaomicron. pRRI4 could be transferred from the colonic Bacteroides species only if the donor strain contained the Tcr Emr 12256 element in its chromosome. These results show that transfer of naturally occurring elements can be demonstrated under laboratory conditions. Evidence that such transfers may actually have occurred in nature came from our finding that the tetracycline resistance (Tcr) gene on the P. ruminicola plasmid pRRI4 hybridized on high-stringency Southern blots with the Tcr gene found on the Bacteroides Tcr elements. The presence of the same gene in such distantly related genera of bacteria is most likely to have occurred as a result of horizontal transfer.     

Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4.

Prevotella ruminicola (formerly Bacteroides ruminicola) is an anaerobic, gram-negative, polysaccharide-degrading bacterium which is found in the rumina of cattle. Since P. ruminicola is thought to make an important contribution to digestion of plant material in rumina, the ability to alter this strain genetically might help improve the efficiency of rumen fermentation. However, previously there has been no way to introduce foreign DNA into P. ruminicola strains. In this study we transferred a shuttle vector, pRDB5, from the colonic species Bacteroides uniformis to P. ruminicola B(1)4. The transfer frequency was 10(-6) to 10(-7) per recipient. pRDB5 contains sequences from pBR328, a cryptic colonic Bacteroides plasmid pB8-51, and a colonic Bacteroides tetracycline resistance (Tcr) gene. pRDB5 was mobilized out of B. uniformis by a self-transmissible Bacteroides chromosomal element designated Tcr Emr 12256. pRDB5 replicated in Escherichia coli as well as in Bacteroides spp. and was also mobilized from E. coli to B. uniformis by using IncP plasmid R751. However, direct transfer from E. coli to P. ruminicola B(1)4 was not detected. Thus, to introduce cloned DNA into P. ruminicola B(1)4, it was necessary first to mobilize the plasmid from E. coli to B. uniformis and then to mobilize the plasmid from B. uniformis to P. ruminicola B(1)4.