Transfer of plasmid-borne resistance from a multiply-resistant Staphylococcus aureus isolate,WBG1022

Staphylococcus aureus isolate, WBG1022, was resistant to penicillin, kanamycin, neomycin, streptomycin, chloramphenicol, trimethoprim, cadmium, and ethidium bromide and harbored plasmids of 34.5, 24.5, 4.4, 3.2, and 2.6 kilobases. The plasmids were transferred in mixed-culture transfer and conjugation experiments. No resistance phenotype was associated with the 2.6-kb plasmid. The 3.2-kb and 4.4-kb plasmids encoded chloramphenicol and streptomycin resistance respectively. The 24.5-kb plasmid, pWBG626, encoded joint resistance to penicillin, kanamycin, neomycin, and ethidium bromide. Resistance to trimethoprim and cadmium were chromosomal. The 34.5-kb plasmid, pWBG661, had no resistance phenotype but was found to be conjugative. It also mobilized the 4.4-kb and 24.5-kb plasmids in WBG1022. Restriction endonuclease analysis of pWBG661 with EcoRI, ClaI, PvuII, and BglII restriction enzymes demonstrated that pWBG661 was identical to two previously isolated S. aureus conjugative plasmids, p WBG620 and pWBG637, that also lack resistance phenotypes.     

Survey of plasmids and resistance factors among veterinary isolates of Salmonella enteritidis in Malaysia

Thirty-five veterinary isolates of Salmonella enteritidis were characterized by their susceptibility to 10 antimicrobial agents and by their plasmid profiles on agarose gel electrophoresis. All were susceptible to carbenicillin, chloramphenicol and nalidixic acid but 89% were resistant to tetracycline. When examined, 91% of the isolates harboured plasmids, with sizes ranging from 9.8 to 60 MDa. However, it was only possible to associate the presence of plasmids with tetracycline resistance; plasmids occurring in 90% of the tetracycline-resistant isolates. In conjugation experiments, with Escherichia coli K12 Nal^r as recipient, the tetracycline resistance in three selected S. enteritidis isolates was observed to transfer at frequencies of 3.0×10^-3 to 1.0×10^-2/donor cell. The concomitant transfer of a 56-MDa or 60-MDa plasmid in these three S. enteritidis isolates was also detected.R. Son. A. Ansary and I. Salmah are with the Department of Genetics and Cellular Biology. University of Malaya, 59100 Kuala Lumpur, Malaysia     

Evaluation of Plasmid Content and Tetracycline Resistance Conjugative Transfer in <Emphasis Type="Italic">Enterococcus italicus</Emphasis> Strains of Dairy Origin

Five Enterococcus italicus strains harbouring tet genes responsible for the tetracycline resistance were subjected to plasmid profile determination studies. For four strains tested the profiles showed between three and six plasmid bands, the size of which ranged between 1.6 and 18.5 kb. Southern hybridization experiments associated tetS and tetK genes with chromosomal DNA in all strains and tetM gene with plasmids of around the same size (18.5 kb) in two of the tested strains. The ability of the new species to transfer tetM gene was studied by transfer experiments with the tetracycline-susceptible recipient strains E. faecalis JH2-2 and OG1RF; mobilization experiments were performed with E. faecalis JH 2-2 harbouring the conjugative plasmid pIP501as helper plasmid. The results obtained show that the new enterococcal species was able to acquire antibiotic resistance by conjugation, but not to transfer its plasmids to other bacteria. Further PCR and hybridization experiments carried out to assess the presence of mobilization sequences also suggest that the tetM plasmid from E. italicus is a non-mobilizable plasmid.     

Conjugal transfer and expression of streptococcal transposons in Clostridium acetobutylicum

The transposon-containing streptococcal plasmids pAM211, pCF10, and pINY1275 have been transferred at high frequency (10^-2–10^-3 per recipient, selecting for tetracycline resistance) to the Gram-positive anaerobe Clostridium acetobutylicum. Selection in the presence of two antibiotics (tetracycline and erythromycin) with the plasmids pAM 180 and pINY1275 yielded only low numbers of transconjugants (10^-8 per recipient). Matings were done by combining liquid and filter mating procedures under anaerobic conditions. No plasmid DNA could be detected in the transconjugants selected on a minimal medium in the presence of tetracycline. DNA-DNA hybridization experiments with restricted chromosomal DNA using biotinylated pAM120::Tn916 as probe revealed the presence of homologous sequences in the transconjugants but not in Clostridium acetobutylicum wild type. The transconjugants were used as donors in mating experiments with tetracycline-sensitive Bacillus subtilis and Streptococcus lactis subspec. diacetylactis. In both cases tetracycline-resistant strains were found. Transfer frequencies in these experiments were less than 10^-7 per recipient.     

Conjugative transfer of tetracycline resistance in rumen streptococcal strains

In 11% of testedStreptococcus bovis strains a conjugative transfer of tetracycline resistance was observed when mating experiments were carried out on membrane filters. The recipient strain used wasS. bovis BM114 with chromosomal resistance to rifampicin. In addition, in two strains tetracycline resistance was transferred also to recipient strainEnterococcus faecium AL6. The transfer frequencies were in the range of 10⁻⁶ to 10⁻³. The donor strains were screened for the presence of plasmids and one up to four bands of plasmid DNA in all tested strains were revealed. In spite of that isolation of plasmid DNA was successful only in 53/4/114 transconjugants. Transconjugant 32/114 contained amylase activity which was higher than in the donor strain.     

A site-specific DNA inversion in Bacteroides plasmid pBF4 is influenced by the presence of the conjugal tetracycline resistance element.

pBF4 is a 42-kb R plasmid from Bacteroides fragilis which transfers clindamycin resistance (Clr) independently of the chromosomal tetracycline resistance (Tcr) transfer element. We have found that this plasmid exists in two nonequimolar conformations, A and B. These forms differ by an inversion of approximately 11.5 kb which does not involve the repeated DNA sequences previously mapped on the plasmid. The presence of chromosomal tetracycline resistance conjugal elements influences the relative amounts of the two conformations: induction with tetracycline shifts the dominant form from B to A.     

Mapping andfi character determination of R factors ofEnterobacter cloacae DF13

In conjugation experiments betweenEnterobacter cloacae DF13 andEscherichia coli K12, resistances against tetracycline, sulfanilamide, streptomycin, and chloramphenicol were nearly always transferred simultaneously. These properties could be transferred fromE. coli exconjugants by transduction to a drug-sensitiveE. coli K12 strain with bacteriophage P1kc. It may be inferred thatEnt. cloacae DF 13 harbours a multiple R factor, which promotes its own transfer. This R factor was found to be of thefi ⁺ type. The molecular nature of this R factor was studied by labelling the DNA of an exconjugant with³H-thymidine, careful lysis, sedimentation of the chromosomal DNA, and characterization of the circular DNA by sucrose-gradient centrifugation, equilibriumdensity centrifugation in CsCl containing ethidium bromide and by electron microscopy. By these methods the multiple R factor was identified as a circular DNA molecule with a contour length of 22.6 Μm, corresponding to a molecular weight of 45 × 10⁶ daltons. A segregant R factor harbouring resistance against tetracycline only, was found to have a contour length of 16.0 Μm and a sedimentation constant of 58 S. In addition to the multiple R factor, the wild-type strain harboured a plasmid with a sedimentation constant of 38 S, corresponding to a molecular weight of 16 × 10⁶ daltons. The function of this plasmid is unknown. After many transfers on agar slants spontaneous segregation of the R factor was observed and several types of segregants were obtained. In most segregants, resistance against streptomycin could not be transferred by conjugation and could not be mobilized by other sex factors. Some of these segregants had acquired a requirement for methionine; in these, the streptomycin-resistance determinant may be integrated into the chromosome. The resistance pattern of the various types of segregants and exconjugants allowed to draw a circular map of the R factor. The order of markers is ---tet---rtf---sul---str---cml-. After short-term conjugation experiments most exconjugants were found to have received resistance against sulfanilamides only. This resistance determinant does not promote its own transfer by conjugation but could be mobilized by other sex factors. An exconjugant become resistant against tetracycline and sulfanilamide, was found to harbour two independent plasmids of which only that carrying resistance against tetracycline promoted its own transfer. Consequently a second R factor, determining resistance against sulfanilamide alone must be present inEnt. cloacae DF13. This R factor was identified as a circular DNA molecule with a sedimentation constant of 26 S, a contour length of 2.6 Μm and a buoyant density of 1.709. From a strain harbouring the independent R(SA) plasmid and an R(TC) fragment of the multiple R factor, transductants resistant against sulfanilamide were obtained. These were found to harbour an R(SA) plasmid with properties of a defective Rfi ⁺ transfer factor. Most probably these plasmids resulted from recombination between the R(SA) plasmid and the Rtf region of the R(TC) fragment. The author published previously under the name of “G. A. Tieze”. The technical assistance of Miss J.T.M.P.A. Havermans, Mrs. A. Mak-Zuidervaart, and Mr. M. V. M. Lafleur is gratefully acknowledged. The authors thank Dr. E. F. J. van Bruggen and Dr. D. Ellens for the electronmicroscopical measurements.     

Molecular analysis of tetracycline resistance in Salmonella enterica subsp. enterica serovars Typhimurium, enteritidis, Dublin, Choleraesuis, Hadar and Saintpaul: construction and application of specific gene probes

A total of 65 epidemiologically unrelated tetracycline-resistant isolates of the six Salmonella enterica subsp. enterica (Salm.) serovars Dublin, Choleraesuis, Typhimurium, Enteritidis, Hadar and Saintpaul were investigated for the presence of tetracycline resistance genes. For this, specific gene probes of the tetracycline resistance genes (tet) of the hybridization classes A, B, C, D, E and G were constructed by cloning PCR-amplified internal segments of the respective tet structural genes. These gene probes were sequenced and used in hybridization experiments with plasmid DNA or endonuclease digested whole cell DNA as targets. Only tet(A) genes were detected on plasmids in all Salm. Dublin isolates as well as in single isolates of Salm. Choleraesuis and Salm. Typhimurium. Genes of the hybridization classes B, C, D and G, but also in some cases those of class A, were located in the chromosomal DNA of the corresponding Salmonella isolates. Restriction fragment length polymorphisms (RFLPs) of tet gene carrying fragments were detected in chromosomally tetracycline-resistant isolates. These RFLPs might represent valuable additional tools for the identification and characterization of tetracycline-resistant Salmonella isolates.     

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.     

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.     

SdrI of Staphylococcus saprophyticus is a multifunctional protein: localization of the fibronectin-binding site

The transferability of a large plasmid that harbors a tetracycline resistance gene tet (S), to fish and human pathogens was assessed using electrotransformation and conjugation. The plasmid, originally isolated from fish intestinal Lactococcus lactis ssp. lactis KYA-7, has potent antagonistic activity against the selected recipients ( Lactococcus garvieae and Listeria monocytogenes ), preventing conjugation. Therefore the tetracycline resistance determinant was transferred via electroporation to L . garvieae . A transformant clone was used as the donor in conjugation experiments with three different L. monocytogenes strains. To our knowledge, this is the first study showing the transfer of an antibiotic resistance plasmid from fish-associated lactic bacteria to L. monocytogenes , even if the donor L. garvieae was not the original host of the tetracycline resistance but experimentally created by electroporation. These results demonstrate that the antibiotic resistance genes in the fish intestinal bacteria have the potential to spread both to fish and human pathogens, posing a risk to aquaculture and consumer safety.     

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.     

Tetracycline-resistance inEscherichia coli; a genetic contribution

A non-transmissible tetracycline-resistance plasmid inE. coli was found to be transmissible by transduction and by conjugation with the aid of theE. coli K12 sex-factor. Transfer of the tetracycline-resistance plasmid (R-tet) by transduction or conjugation to anE. coli K12 Hfr strain revealed that the plasmid was incompatible with the integrated F-factor. Selection for tetracycline-resistance after conjugation or transduction yielded Hfr colonies which carried the tetracycline-resistance determinant as a chromosomal marker. The tetracycline-resistance determinant was integrated at the 1 min region of theE. coli chromosome map (Taylor and Trotter, 1967) between the markersara andleu. Apart from Hfr colonies with a chromosomal tetracycline-resistance determinant, F-gal⁺-mediated transfer of R-tet to strain Hfr R4 gave some colonies in which the tetracycline-resistance determinant was carried on a fused plasmid that, besides the resistance determinant, contained thegal ⁺ marker of the original F-gal ⁺. This fused plasmid is transmissible and confers to an F⁻ cell male-specific phage-sensitivity, like an F-factor does. It is suggested that this fused plasmid, which is compatible with the integrated F-factor in the Hfr R4 cells, arose by recombination between F-gal ⁺ and R-tet.     

Characterization and transferability of Clostridium perfringens plasmids.

Two strains of Clostridium perfringens resistant to clindamycin (Cl), chloramphenicol (Cm), erythromycin (Em), and tetracycline (Tc) were isolated in France in 1974 and 1975. For one of these strains, curing experiments and molecular characterization of the extrachromosomal DNA clearly demonstrate the existence of two plasmids, plP401 (54 kilobases) and plP402 (63 kilobases), which, respectively, code for Tc Cm and Em Cl resistance. With mixed cultures, the Tc Cm plasmid is transferable to sensitive strains of C. perfringens; a segregation of these markers is frequently observed during mating experiments. In contrast, the transfer of the naturally occurring plasmid Em Cl does not occur at a significant rate. In performing transfer experiments in axenic mice, we obtained a Cl^r Em^r Tc^r transcipient whose chromosomal properties are those of a hybrid. When used in mating as a parental strain, this strain promotes chromosomal gene exchange. The role of the plasmid in this phenomenon is discussed, these transcipients being generally Cl^r Em^r Tc^r. The plasmid transfer is not limited to antibiotic resistance plasmids, the transferability of a bacteriocinogenic plasmid, plP404, harbored by C. perfringens BP6K-N5 being shown also. The transfer mechanism remains to be proved; it might be a conjugation process, a cell-to-cell contact being necessary for the transfer.     

Evidence for natural horizontal transfer of tetO gene between Campylobacter jejuni strains in chickens

AIMS: The transfer of tetO gene conferring resistance to tetracycline was studied between Campylobacter jejuni strains, in the digestive tract of chickens. METHODS AND RESULTS: In vitro conjugation experiments were first performed in order to select donor/recipient couples for further in vivo assay. Then, chickens were inoculated with a donor/recipient couple of C. jejuni strains displaying spontaneous in vitro tetracycline resistance gene transfer. The donor was a tetracycline-resistant ampicillin-susceptible strain, and the recipient was a tetracycline-susceptible ampicillin-resistant strain. Chicken droppings were streaked on antimicrobial selective media and bi-resistant Campylobacter isolates were further characterized according to their donor or recipient flaA gene RFLP profile. The acquisition of tetracycline-resistance gene by the recipient C. jejuni strain from the donor C. jejuni strain was confirmed by tetO PCR. CONCLUSIONS: The study showed that transfer of tetO gene occurs rapidly and without antimicrobial selection pressure between C. jejuni strains in the digestive tract of chickens. SIGNIFICANCE AND IMPACT OF THE STUDY: The rapid and spontaneous transfer of tetO gene may explain the high prevalence of tetracycline resistance in chicken Campylobacter strains.     

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.     

Identification of a ChromosomalShigella flexneriMulti-Antibiotic Resistance Locus Which Shares Sequence and Organizational Similarity with the Resistance Region of the Plasmid NR1

The ampicillin resistance gene fromShigella flexneri2a strain YSH6000 was cloned and shown by Southern hybridization analysis to be closely linked to the previously cloned streptomycin, chloramphenicol, and tetracycline resistance determinants, which are borne on a chromosomally integrated 99-kb element. Analysis of this chromosomal multi-antibiotic resistance locus revealed that it had a high level of sequence and organizational similarity to an equivalent region of theShigellaR-plasmid, NR1. However, the chromosomal locus exhibited several differences, including the presence of two stretches of sequence derived from IS elements, the precise insertion of a β-lactamase encodingoxa1cassette into the Tn21-borne integron In2, a possible 17.5-kb deletion, and the loss or inactivation of the mercury resistance determinant. Based on these data, it is proposed that the chromosomal locus arose following integration of an NR1-like plasmid.     

Clustering of mutations affecting alginic acid biosynthesis in mucoid Pseudomonas aeruginosa.

A 10-kilobase DNA fragment previously shown to contain the phosphomannose isomerase gene (pmi) of Pseudomonas aeruginosa was used to construct a pBR325-based hybrid that can be propagated in P. aeruginosa only by the formation of a chromosomal-plasmid cointegrate. This plasmid, designated pAD4008, was inserted into the P. aeruginosa chromosome by recombination at a site of homology between the cloned P. aeruginosa DNA and the chromosome. Mobilization of pAD4008 into P. aeruginosa PAO and 8830 and selection for the stable acquisition of tetracycline resistance resulted in specific and predictable changes in the pattern of endonuclease restriction sites in the phosphomannose isomerase gene region of the chromosomes. Chromosomal DNA from the tetracycline-resistant transformants was used to clone the drug resistance determinant with Bg/II or XbaI, thereby allowing the "walking" of the P. aeruginosa chromosome in the vicinity of the pmi gene. Analysis of overlapping tetracycline-resistant clones indicated the presence of sequences homologous to the DNA insert of plasmid pAD2, a recombinant clone of P. aeruginosa origin previously shown to complement several alginate-negative mutants. Restriction mapping, subcloning, and complementation analysis of a 30-kilobase DNA region demonstrated the tight clustering of several genetic loci involved in alginate biosynthesis. Furthermore, the tetracycline resistance determinant in PAO strain transformed by pAD4008 was mapped on the chromosome by plasmid FP2-mediated conjugation and was found to be located near 45 min.     

Molecular nature of two Haemophilus influenzae R factors containing resistances and the multiple integration of drug resistance transposons.

The 36-megadalton Haemophilus influenzae R plasmid pHK539 was found to specify resistance to tetracycline (Tc) and ampicillin (Ap). It was shown by molecular hybridization studies and by electron microscopy that the plasmid pHK539 contained the tetracycline translocation deoxyribonucleic acid (DNA)segment (TnTc) as well as the ampicillin translocation segment (TnAp). The TnAp was integrated in the stem of TnTc. The 34-megadalton H. influenzae R plasmid pRI234 carried a translocatable DNA segment which specified both tetracycline and chloramphenicol (Cm) resistance. Self-annealing and DNA-DNA heteroduplex experiments indicated that this transposon is probably composed of TnTc containing an insertion of a chloramphenicol resistance transposon (TnCm). TnCm is inserted into one of the components of the TnTc inverted repetitions and is itself flanked on both sides by long inverted repetitions. The H. influenzae plasmids pHK539 and pRI234 had more than 60% of their polynucleotide sequences in common with all the other 30- to 40-megadalton R factors recently found in H. influenzae isolates from different countries. The tetracycline-chloramphenicol resistance transposon of pRI234 was integrated twice at different sites in the plasmid after its growth in medium containing tetracycline. The presence of the two copies of the transposon was correlated with higher minimum inhibitory concentrations against tetracycline as well as against chloramphenicol. After its growth in medium containing tetracycline, the H. influenzae R plasmid pFR16017 specifying Tc resistance contained one, two, three, or even four copies of TnTc integrated at different sites in the plasmid, or the loop of TnTc was amplified. The heterogeneity of the pFR16017 plasmid was seen in all single-colony isolates and correlated with a higher minimum inhibitory concentration against tetracycline.     

Chromosomal integration of plasmid DNA by homologous recombination in Enterococcus faecalis and Lactococcus lactis subsp. lactis hosts harboring Tn919.

Integration of pCI192, a pBR322-derived vector plasmid containing homology to the chromosomally located conjugative transposon Tn919 was observed in two strains that harbor Tn919, namely, Enterococcus faecalis GF590 and Lactococcus lactis subsp. lactis CH919. Hybridization analysis indicated that single-copy integration of the plasmid had occurred at low frequency. The Tn919::plasmid structure was conjugated from an E. faecalis donor to a L. lactis recipient, although at lower frequencies than was Tn919. Segregation of the tetracycline and chloramphenicol resistance markers during conjugation was observed. The integration strategy described allows for DNA manipulations to be performed in an easily manipulated model host strain with the subsequent transfer of integrated structures by conjugation to any strain capable of receiving Tn919. The results indicate that homologous recombination events may be used to introduce plasmid-encoded genes to the lactococcal chromosome.