Survival of enterococci and Tn916-like conjugative transposons in soil

The persistence of Enterococcus faecalis, fecal enterococci from swine waste, and Tn916-like elements was determined following inoculation into autoclaved and native soil microcosms. When cells of E. faecalis CG110 (Tn916) were inoculated into native microcosms, enterococcal viability in the soil decreased approximately 5 orders of magnitude (4.8 x 10(5) CFU/g soil to < 10 CFU/g) after 5 weeks. In autoclaved microcosms, the viability of E. faecalis decreased by only 20% in 5 weeks. In contrast, the content of Tn916, based on PCR of DNA extracts from soil microcosms, decreased by about 20% in both native and autoclaved microcosms. Similar results were obtained when the source of fecal enterococci and Tn916-like elements was swine waste. Because the concentration of Tn916-independent E. faecalis DNA (the D-alanine D-alanine ligase gene), based on PCR, decreased to nearly undetectable levels (at least 3 orders of magnitude) after 5 weeks in the native microcosms, the evidence suggests Tn916 stability in the soil results from en masse transfer of the transposon to the normal soil microflora and not survival of E. faecalis DNA in the soil system. Results from denaturing gradient gel electrophoresis suggest that multiple forms of Tn916 occur in swine waste, but only forms most like Tn916 exhibit stability in the soil.     

Introduction of the Streptococcus faecalis transposon Tn916 into Bacillus thuringiensis subsp. israelensis

The conjugative Streptococcus faecalis transposon Tn916 was introduced into Bacillus thuringiensis subsp. israelensis by filter matings with S. faecalis. B. thuringiensis transconjugants resistant to tetracycline (Tetr) were detected at a frequency of approximately 7.0 X 10(-7) per recipient cell during filter matings, whereas transfer of Tn916 was not observed in broth matings. The Tetr phenotype in subsp. israelensis was stable in the absence of antibiotic selection. Southern hybridization analysis revealed that Tn916 had inserted into several different sites on the B. thuringiensis subsp. israelensis chromosome but insertion into plasmid DNA was not observed. Movement of Tn916 was demonstrated when Tetr B. thuringiensis transconjugants were mated with isogenic recipients. Southern hybridizations, however, showed that the resulting Tetr isolates contained Tn916 junction fragments that were nearly identical to the donor, suggesting that this movement resulted from transfer of chromosomal DNA from donor to recipient or from a fusion of mating cells, rather than conjugative transposition of the Tn element.     

Tn916-dependent conjugal transfer of PC194 and PUB110 from Bacillus subtilis into Bacillus thuringiensis subsp. israelensis

The Staphylococcus aureus plasmids pC194 and pUB110 were introduced into Bacillus thuringiensis subsp. israelensis by using the Streptococcus faecalis transposon Tn916 as a mobilizing agent. Plasmid transfer occurred only when B. thuringiensis subsp. israelensis was mated with a B. subtilis donor that contained both pC194 and pUB110 and Tn916; plasmid transfer was not observed in the absence of the transposon. B. thuringiensis transconjugants resistant to chloramphenicol (Cmr) and tetracycline (Tetr) were detected at a frequency of 1.96 x 10(-6) per recipient cell, whereas the Tetr phenotype, but not the Cmr, was observed at a frequency of 1.09 x 10(-4). The converse, Cmr but not Tetr, was observed at a frequency of 2.94 X 10(-5). The transfer of pUB110 from B. subtilis to B. thuringiensis subsp. israelensis was observed at a frequency of 3.0 x 10(-6) per recipient cell but concomitant transfer of pUB110 and Tn916 was not observed. Mobilization of plasmid pE194 was not observed under these conditions. Transconjugants were detected in filter matings only, not in broth. The Tn916 phenotype was maintained during serial passage of B. thuringiensis without selection, whereas the pC194 phenotype was not. Unlike pC194, however, pUB110 remained stable in B. thuringiensis during several passages through nonselective medium. Southern hybridization analysis demonstrated that Tn916 had inserted into several different sites on the B. thuringiensis chromosome and that pC194 and pUB110 were maintained as an autonomous plasmid.     

Tetracycline enhances Tn916-mediated conjugal transfer.

Pregrowth of the donor on medium containing tetracycline increased conjugative transposition of Tn916 and the transposon-dependent mobilization of pC194 19- to 119-fold in matings between Bacillus subtilis and Bacillus thuringiensis subsp. israelensis. Tn916 and pC194 transferred independently under these conditions. When Enterococcus faecalis was the donor and B. thuringiensis subsp. israelensis the recipient, pregrowth in tetracycline increased the conjugative transposition frequency by approximately 15-fold. Tetracycline-enhanced conjugation appeared during matings as short as 3 h in length. Pregrowth in tetracycline did not enhance conjugation in Bacillus sphaericus x B. thuringiensis subsp. israelensis or B. thuringiensis subsp. israelensis x B. subtilis matings. Incorporation of tetracycline into the mating medium, at concentrations that did not inhibit growth of the B. thuringiensis subsp. israelensis recipient, resulted in conjugation frequencies similar to those obtained by pregrowth of the B. subtilis donors in antibiotic-containing medium. The data suggest stimulation of donor function by tetracycline.     

Insertion of Tn916 into Bacillus pumilus plasmid pMGD302 and evidence for plasmid transfer by conjugation.

As part of an effort to develop systems for genetic analysis of strains of Bacillus pumilus which are being used as a microbial hay preservative, we introduced the conjugative Enterococcus faecalis transposon Tn916 into B. pumilus ATCC 1 and two naturally occurring hay isolates of B. pumilus. B. pumilus transconjugants resistant to tetracycline were detected at a frequency of approximately 6.5 x 10(-7) per recipient after filter mating with E. faecalis CG110. Southern hybridization confirmed the insertion of Tn916 into several different sites in the B. pumilus chromosome. Transfer of Tn916 also was observed between strains of B. pumilus in filter matings, and one donor strain transferred tetracycline resistance to recipients in broth matings at high frequency (up to 3.4 x 10(-5) per recipient). Transfer from this donor strain in broth matings was DNase-resistant and was not mediated by culture filtrates. Transconjugants from these broth matings contained derivatives of a cryptic plasmid (pMGD302, approx 60 kb) from the donor strain with Tn916 inserted at various sites. The plasmids containing Tn916 insertions transferred to a B. pumilus recipient strain at frequencies of approx 5 x 10(-6) per recipient. This evidence suggests that pMGD302 can transfer by a process resembling conjugation between strains of B. pumilus.     

Tn5381, a conjugative transposon identifiable as a circular form in Enterococcus faecalis.

We have identified two 19-kb conjugative transposons (Tn5381 and Tn5383) in separate strains of multiply resistant Enterococcus faecalis. These transposons confer resistance to tetracycline and minocycline via a tetM gene, are capable of both chromosomal and plasmid integration in a Rec- environment, and transfer between strains in the absence of detectable plasmid DNA at frequencies ranging from < 1 x 10(-9) to 2 x 10(-5) per donor CFU, depending on the donor strain and the growth conditions. Hybridization studies indicate that these transposons are closely related to Tn916. We have identified bands of ca. 19 kb on agarose gel separations of alkaline lysis preparations from E. faecalis strains containing chromosomal copies of Tn5381, which we have confirmed to be a circularized form of this transposon. This phenomenon has previously been observed only when Tn916 has been cloned in Escherichia coli. Overnight growth of donor strains in the presence of subinhibitory concentrations of tetracycline results in an approximately 10-fold increase in transfer frequency of Tn5381 into enterococcal recipients and an increase in the amount of the circular form of Tn5381 as detectable by hybridization. These results suggest that Tn5381 is a Tn916-related conjugative transposon for which the appearance of a circular form and the conjugative-transfer frequency are regulated by a mechanism(s) affected by the presence of tetracycline in the growth medium.     

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon,in the Bacillus cereus group

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.     

Analysis of the Requirement for a pUB110 mob Region during Tn916-Dependent Mobilization

Tn916-dependent mobilization of nonconjugative plasmids pUB110 and its derivative pUB110Deltam was compared. Deleting a 787-bp fragment from the pUB110 mob region created plasmid pUB110Deltam. Deletion of the mob region of pUB110 rendered the plasmid nontransferable by the conjugative plasmids of Bacillus thuringiensis subsp. israelensis. During matings between Bacillus subtilis (Tn916) and B. thuringiensis subsp. israelensis, however, Tn916-dependent mobilization of plasmids pUB110 and pUB110Deltam was observed at a frequency of approximately 2 x 10(-6) transconjugants per donor. The results show that Tn916-mediated conjugal transfer of plasmids is a mob-independent event. Jaworski and Clewell (J. Bacteriol 177; 6644-6651) recently demonstrated the presence of an IncP-like nicking site in the oriT of Tn916. These data suggest that a IncP-like nickling site is essential for Tn916-mediated plasmid transfer.     

Ecology of antibiotic resistance genes: characterization of enterococci from houseflies collected in food settings

In this project, enterococci from the digestive tracts of 260 houseflies (Musca domestica L.) collected from five restaurants were characterized. Houseflies frequently (97% of the flies were positive) carried enterococci (mean, 3.1 x 10(3) CFU/fly). Using multiplex PCR, 205 of 355 randomly selected enterococcal isolates were identified and characterized. The majority of these isolates were Enterococcus faecalis (88.2%); in addition, 6.8% were E. faecium, and 4.9% were E. casseliflavus. E. faecalis isolates were phenotypically resistant to tetracycline (66.3%), erythromycin (23.8%), streptomycin (11.6%), ciprofloxacin (9.9%), and kanamycin (8.3%). Tetracycline resistance in E. faecalis was encoded by tet(M) (65.8%), tet(O) (1.7%), and tet(W) (0.8%). The majority (78.3%) of the erythromycin-resistant E. faecalis isolates carried erm(B). The conjugative transposon Tn916 and members of the Tn916/Tn1545 family were detected in 30.2% and 34.6% of the identified isolates, respectively. E. faecalis carried virulence genes, including a gelatinase gene (gelE; 70.7%), an aggregation substance gene (asa1; 33.2%), an enterococcus surface protein gene (esp; 8.8%), and a cytolysin gene (cylA; 8.8%). Phenotypic assays showed that 91.4% of the isolates with the gelE gene were gelatinolytic and that 46.7% of the isolates with the asa1 gene aggregated. All isolates with the cylA gene were hemolytic on human blood. This study showed that houseflies in food-handling and -serving facilities carry antibiotic-resistant and potentially virulent enterococci that have the capacity for horizontal transfer of antibiotic resistance genes to other bacteria.     

Two conjugation systems associated with Streptococcus faecalis plasmid pCF10: identification of a conjugative transposon that transfers between S. faecalis and Bacillus subtilis.

The tetracycline resistance plasmid pCF10 (58 kilobases [kb]) of Streptococcus faecalis possesses two separate conjugation systems. A 25-kb region of the plasmid (designated TRA) was shown previously to determine pheromone response and conjugation functions required for transfer of pCF10 between S. faecalis cells (P. J. Christie and G. M. Dunny, Plasmid 15:230-241, 1986). When S. faecalis cells were mixed with Bacillus subtilis in broth, tetracycline resistance was transferred from S. faecalis. The tetracycline-resistant B. subtilis cells contained a 16-kb region of pCF10 (distinct from TRA) that carried the tetracycline resistance determinant (Tetr). This Tetr element was found to transfer between S. faecalis and B. subtilis strains in the absence of plasmids. Genetic and molecular techniques were used to establish locations of the element at several different sites on the B. subtilis chromosome. The Tetr element could be transferred in filter matings from B. subtilis to S. faecalis strains and between recombination-proficient and -deficient S. faecalis strains in the absence of any plasmid DNA. The transfer required direct cell-to-cell contact and was not inhibited by DNase. The Tetr element was shown to transpose from the S. faecalis chromosome to various locations within the hemolysin plasmid pAD1. Together, the data indicate that the Tetr element, termed transposon Tn925, is very similar to the conjugative transposon Tn916 in both structure and function. A derivative of Tn925, containing transposon Tn917 inserted into a site approximately 3 kb from one end, exhibited elevated transfer frequencies and may provide a useful means for delivering Tn917 by conjugation into various gram-positive species.     

Introduction of transposon Tn916 DNA into Haemophilus influenzae and Haemophilus parainfluenzae.

Enterococcus (Streptococcus) faecalis transposon Tn916 was introduced into Haemophilus influenzae Rd and Haemophilus parainfluenzae by transformation and demonstrated to transpose efficiently. Haemophilus transformants resistant to tetracycline were observed at a frequency of approximately 3 x 10(2) to 5 x 10(3)/micrograms of either pAM120 (pGL101::Tn916) or pAM180 (pAM81::Tn916) plasmid DNAs, which are incapable of autonomous replication in this host. Restriction enzyme analysis and Southern blot hybridization revealed that (i) Tn916 integrates into many different sites in the H. influenzae and H. parainfluenzae genomes; (ii) only the 16.4-kilobase-pair Tn916 DNA integrates, and no vector DNA was detected; and (iii) the Tetr phenotype was stable in the absence of selective pressure. Second-generation Tn916 transformants occurred at the high frequency of chromosomal markers and retained their original chromosomal locations. Similar results were obtained with H. influenzae Rd BC200 rec-1 as the recipient strain, which suggests host rec functions are not required in Tn916 integrative transposition. Transposition with Tn916 is an important procedure for mutagenesis of Haemophilus species.     

Characterization of transferable tetracycline resistance genes in Enterococcus faecalis isolated from raw food

The prevalence of tetracycline resistance, and of specific genetic determinants for this resistance was investigated in 1003 strains of Enterococcus faecalis isolated from various raw food products originating from five categories including chicken meat, other poultry meat, beef, pork, and 'other'. For the 238 resistant isolates identified, the ability to transfer the resistant phenotype to a given recipient in vitro was investigated. New and interesting observations were that the tet(L) resistance determinant was more readily transferred than tet(M), and that the presence of Tn916-like elements known to encode tet(M) did not correlate with increased transferability of the resistant phenotype.     

Involvement of Tn4430 in transfer of Bacillus anthracis plasmids mediated by Bacillus thuringiensis plasmid pXO12.

The self-transmissible plasmid pXO12 (112.5 kilobases [kb]), originally isolated from strain 4042A of Bacillus thuringiensis subsp. thuringiensis, codes for production of the insecticidal crystal protein (Cry+). The mechanism of pXO12-mediated plasmid transfer was investigated by monitoring the cotransfer of the tetracycline resistance plasmid pBC16 (4.2 kb) and the Bacillus anthracis toxin and capsule plasmids, pXO1 (168 kb) and pXO2 (85.6 kb), respectively. In matings of B. anthracis donors with B. anthracis and Bacillus cereus recipients, the number of Tcr transcipients ranged from 4.8 x 10(4) to 3.9 x 10(6)/ml (frequencies ranged from 1.6 x 10(-4) to 7.1 x 10(-2), and 0.3 to 0.4% of them simultaneously inherited pXO1 or pXO2. Physical analysis of the transferred plasmids suggested that pBC16 was transferred by the process of donation and that the large B. anthracis plasmids were transferred by the process of conduction. The transfer of pXO1 and pXO2 involved the transposition of Tn4430 from pXO12 onto these plasmids. DNA-DNA hybridization experiments demonstrated that Tn4430 was located on a 16.0-kb AvaI fragment of pXO12. Examination of Tra- and Cry- derivatives of pXO12 showed that this fragment also harbored information involved in crystal formation and was adjacent to a restriction fragment containing DNA sequences carrying information required for conjugal transfer.     

Introduction of Tn916 and pAM beta 1 into Streptococcus bovis JB1 by conjugation.

The transposon Tn916 and self-mobilizing plasmid pAM beta 1 were conjugated from Enterococcus faecalis to the ruminal bacterium Streptococcus bovis JB1. Transconjugants were identified by resistance to tetracycline (Tn916) or erythromycin (pAM beta 1) and by Southern hybridization analyses. Transfer frequencies were 7.0 x 10(-6) and 1.0 x 10(-6) per recipient cell for Tn916 and pAM beta 1, respectively. The transconjugants JB1/Tn916 and JB1/pAM beta 1 were used as donors for matings with E. faecalis, Bacillus subtilis, and the ruminal bacterium Butyrivibrio fibrisolvens. While pAM beta 1 was successfully transferred to all three organisms, Tn916 was transferred only into B. subtilis and B. fibrisolvens at very low frequencies. This is the first report of conjugal DNA transfers between two ruminal organisms.     

Natural transfer of conjugative transposon Tn916 between gram-positive and gram-negative bacteria.

The conjugative streptococcal transposon Tn916 was found to transfer naturally between a variety of gram-positive and gram-negative eubacteria. Enterococcus faecalis hosting the transposon could serve as a donor for Alcaligenes eutrophus, Citrobacter freundii, and Escherichia coli at frequencies of 10(-6) to 10(-8). No transfer was observed with several phototrophic species. Mating of an E. coli strain carrying Tn916 yielded transconjugants with Bacillus subtilis, Clostridium acetobutylicum, Enterococcus faecalis, and Streptococcus lactis subsp. diacetylactis at frequencies of 10(-4) to 10(-6). Acetobacterium woodii was the only gram-positive organism tested that did not accept the transposon from a gram-negative donor. The results prove the ability of conjugative transposable elements such as Tn916 for natural cross-species gene transfer, thus potentially contributing to bacterial evolution.     

Introduction of Tn916 and pAM beta 1 into Streptococcus bovis JB1 by conjugation.

The transposon Tn916 and self-mobilizing plasmid pAM beta 1 were conjugated from Enterococcus faecalis to the ruminal bacterium Streptococcus bovis JB1. Transconjugants were identified by resistance to tetracycline (Tn916) or erythromycin (pAM beta 1) and by Southern hybridization analyses. Transfer frequencies were 7.0 x 10(-6) and 1.0 x 10(-6) per recipient cell for Tn916 and pAM beta 1, respectively. The transconjugants JB1/Tn916 and JB1/pAM beta 1 were used as donors for matings with E. faecalis, Bacillus subtilis, and the ruminal bacterium Butyrivibrio fibrisolvens. While pAM beta 1 was successfully transferred to all three organisms, Tn916 was transferred only into B. subtilis and B. fibrisolvens at very low frequencies. This is the first report of conjugal DNA transfers between two ruminal organisms.     

Occurrence, structure, and mobility of Tn1546-like elements in environmental isolates of vancomycin-resistant enterococci

The occurrence, structure, and mobility of Tn1546-like elements were studied in environmental vancomycin-resistant enterococci (VRE) isolated from municipal sewage, activated sludge, pharmaceutical waste derived from antibiotic production, seawater, blue mussels, and soil. Of 200 presumptive VRE isolates tested, 71 (35%) harbored vanA. Pulsed-field gel electrophoresis analysis allowed the detection of 26 subtypes, which were identified as Enterococcus faecium (n = 13), E. casseliflavus (n = 6), E. mundtii (n = 3), E. faecalis (n = 3), and E. durans (n = 1) by phenotypic tests and 16S ribosomal DNA sequencing. Long PCR-restriction fragment length polymorphism (L-PCR-RFLP) analysis of Tn1546-like elements and PCR analysis of internal regions revealed the presence of seven groups among the 29 strains studied. The most common group (group 1) corresponded to the structure of Tn1546 in the prototype strain E. faecium BM4147. Two novel L-PCR-RFLP patterns (groups 3 and 4) were found for E. casseliflavus strains. Indistinguishable Tn1546-like elements occurred in VRE strains belonging to different species or originating from different sources. Interspecies plasmid-mediated transfer of vancomycin resistance to E. faecium BM4105 was demonstrated for E. faecalis, E. mundtii, and E. durans. This study indicates that VRE, including species other than E. faecium and E. faecalis, are widespread in nature and in environments that are not exposed to vancomycin selection and not heavily contaminated with feces, such as seawater, blue mussels, and nonagricultural soil. Tn1546-like elements can readily transfer between enterococci of different species and ecological origins, therefore raising questions about the origin of these transposable elements and their possible transfer between environmental and clinical settings.     

Characterization of Tn1546, a Tn3-related transposon conferring glycopeptide resistance by synthesis of depsipeptide peptidoglycan precursors in Enterococcus faecium BM4147.

Sequence determination of the flanking regions of the vancomycin resistance van gene cluster carried by pIP816 in Enterococcus faecium BM4147 revealed similarity to transposons of the Tn3 family. Imperfect inverted repeats (36 of 38 bp) delineated a 10,851-bp element designated Tn1546. The 4-kb region located upstream from the vanR gene contained two open reading frames (ORF) transcribed in opposite directions. The deduced amino acid sequence of ORF1 (988 residues) displayed, respectively, 56 and 42% identity to those of the transposases of Tn4430 from Bacillus thuringiensis and of Tn917 from Enterococcus faecalis. The product of ORF2 (191 residues) was related to the resolvase of Tn917 (33% amino acid identity) and to the Res protein (48%) of plasmid pIP404 from Clostridium perfringens. Tn1546 transposed consecutively in Escherichia coli from plasmid pUC18 into pOX38 and from pOX38 into various sites of pBR329. Transposition was replicative, led to the formation of cointegrates, and produced a 5-bp duplication at the target site. Southern hybridization and DNA amplification revealed the presence of Tn1546-related elements in enterococci highly resistant to glycopeptides. Analysis of sequences surrounding these elements indicated that transposition plays a role in dissemination of the van gene cluster among replicons of human clinical isolates of E. faecium.     

Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani

We report that the streptococcal resistance transposon, Tn916, is conjugally transferred to Clostridium tetani (Utrecht) in intergenic matings. Streptococcus faecalis CG180, harboring a 41-kb plasmid (pAM180) containing Tn916 (15 kb), transferred the transposon-associated tetracycline resistance (Tcr) to C. tetani in filter matings at a frequency of about 10(-4)/donor. An erythromycin resistance marker carried by pAM180 was not transferred, indicating lack of plasmid conjugation or stable inheritance of plasmid sequences. DNA extracted from C. tetani transconjugants was probed with radiolabeled Tn916 using Southern blot analysis and these results indicated that the transposon integrated at multiple host genomic sites. Tn916-carrying C. tetani strains were able to transfer Tcr to suitable recipient strains of C. tetani as well as to S. faecalis recipients. These results indicate that this transposon is able to be disseminated and expressed in obligately anaerobic gram-positive bacteria. Moreover, this system opens avenues for the implementation of transposon mutagenesis in this important pathogenic species.