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.     

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.     

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.     

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.     

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.     

Conjugative Botulinum Neurotoxin-Encoding Plasmids in Clostridium botulinum

Background

Clostridium botulinum produces seven distinct serotypes of botulinum neurotoxins (BoNTs). The genes encoding different subtype neurotoxins of serotypes A, B, F and several dual neurotoxin-producing strains have been shown to reside on plasmids, suggesting that intra- and interspecies transfer of BoNT-encoding plasmids may occur. The objective of the present study was to determine whether these C. botulinum BoNT-encoding plasmids are conjugative.

Methodology/Principal Findings

C. botulinum BoNT-encoding plasmids pBotCDC-A3 (strain CDC-A3), pCLJ (strain 657Ba) and pCLL (strain Eklund 17B) were tagged with the erythromycin resistance marker (Erm) using the ClosTron mutagenesis system by inserting a group II intron into the neurotoxin genes carried on these plasmids. Transfer of the tagged plasmids from the donor strains CDC-A3, 657Ba and Eklund 17B to tetracycline-resistant recipient C. botulinum strains was evaluated in mating experiments. Erythromycin and tetracycline resistant transconjugants were isolated from donor∶recipient mating pairs tested. Transfer of the plasmids to the transconjugants was confirmed by pulsed-field gel electrophoresis (PFGE) and Southern hybridizations. Transfer required cell-to-cell contact and was DNase resistant. This indicates that transfer of these plasmids occurs via a conjugation mechanism.

Conclusions/Significance

This is the first evidence supporting conjugal transfer of native botulinum neurotoxin-encoding plasmids in C. botulinum, and provides a probable mechanism for the lateral distribution of BoNT-encoding plasmids to other C. botulinum strains. The potential transfer of C. botulinum BoNT-encoding plasmids to other bacterial hosts in the environment or within the human intestine is of great concern for human pathogenicity and necessitates further characterization of these plasmids.     

Transferable tetracycline resistance in Clostridium perfringens strains of porcine origin

These studies represent the first systematic survey of the incidence of conjugative antibiotic resistance in Clostridium perfringens. Ninety-two antibiotic-resistant porcine strains were examined to see if they could donate their antibiotic-resistance determinants to sensitive recipient strains. Fifteen of the 89 tetracycline-resistant strains transferred their tetracycline resistance in mixed-plate mating experiments but no transfer of macrolide-lincosamide resistance was detected. The efficiencies of transfer of tetracycline resistance varied from 1.3 X 10(-3) to 1.9 X 10(-6) transconjugants per donor cell. Significantly higher transfer efficiencies were observed when both the donor and recipient strains were derivatives of strain CW 362. These values ranged from 3.7 X 10(-1) to 4.6 X 10(-2) transconjugants per donor cell. This high frequency transfer system should prove invaluable for further genetic studies on this microorganism.     

Identification of a transferable tetracycline resistance plasmid (pCW3) from Clostridium perfringens

A tetracycline- and chloramphenicol-resistant strain of Clostridium perfringens, CW92, was shown to carry two plasmids, pCW2 and pCW3. Twenty-four independently derived tetracycline-sensitive mutants were isolated using a variety of curing agents. All were missing pCW3 but still carried pCW2. Tetracycline resistance could be transferred to a sensitive recipient strain by what appears to be a conjugation-like process. The efficiency of transfer was 2.8 × 10⁻⁵ transcipients per viable donor cell after a 20-h mating. The transcipients transferred tetracycline resistance at a similar frequency. Ten independently derived tetracycline-resistant transcipients all carried pCW3 as shown by agarose gel electrophoresis. The identity of this plasmid in one of these strains was confirmed by electron microscopy and restriction endonuclease analysis. Therefore, pCW3 (30.6 megadaltons) is a transferable tetracycline-resistance plasmid. No chloramphenicol-sensitive mutants or chloramphenicol-resistant transcipients were isolated. Therefore, pCW2 (36.4 megadaltons) remains cryptic.     

Characterisation and transfer of antibiotic resistance genes from enterococci isolated from food

The genetic determinants responsible for the resistances against the antibiotics tetracycline [tet(M), tet(O), tet(S), tet(K) and tet(L)], erythromycin (ermA,B,C; mefA,E; msrA/B; and ereA,B) and chloramphenicol (cat) of 38 antibiotic-resistant Enterococcus faecium and Enterococcus faecalis strains from food were characterised. In addition, the transferability of resistance genes was also assessed using filter mating assays. The tet(L) determinant was the most commonly detected among tetracycline-resistant enterococci (94% of the strains), followed by the tet(M) gene, which occurred in 63.0% of the strains. Tet(K) occurred in 56.0% of the resistant strains, while genes for tet(O) and tet(S) could not be detected. The integrase gene of the Tn916-1545 family of transposons was present in 81.3% of the tetracycline resistant strains, indicating that resistance genes might be transferable by transposons. All chloramphenicol-resistant strains carried a cat gene. 81.8% of the erythromycin-resistant strains carried the ermB gene. Two (9.5%) of the 21 erythromycin-resistant strains, which did not contain ermA,B,C, ereA,B and mphA genes harboured the msrC gene encoding an erythromycin efflux pump, which was confirmed by sequencing the PCR amplicon. In addition, all E. faecium strains contained the msrC gene, but none of the E. faecalis strains. Transfer of the genetic determinants for antibiotic resistance could only be demonstrated in one filter mating experiment, where both the tet(M) and tet(L) genes were transferred from E. faecalis FAIR-E 315 to the E. faecalis OG1X recipient strain. Our results show the presence of various types of resistance genes as well as transposon integrase genes associated with transferable resistances in enterococci, indicating a potential for gene transfer in the food environment.     

Occurrence of R-plasmids in porcine faecal waste and comparison of their transfer rates in laboratory mating systems and anaerobic digesters

Summary Most of the coliform bacteria (77–100%) isolated from porcine faecal waste were resistant to 6 antibiotics. In up to 51% of cases, the genetic determinants for tetracycline, streptomycin and ampicillin resistances were found to be transmissible to a laboratory strain ofEscherichia coli in laboratory mating techniques. Transfer frequency varied according to the mating technique employed. Transfer of resistance determinants during treatment of the faecal waste in a laboratory-scale anaerobic digester was detected erratically in very few cases.     

Drug resistance of Enterococcus faecium clinical isolates and the conjugative transfer of gentamicin and erythromycin resistance traits

Drug resistance and the transferability of resistance were examined in 218 Enterococcus faecium clinical isolates obtained from in-patients of a Japanese university hospital between 1990 and 1999. One hundred and sixty one isolates (73.9%) were drug-resistant and 127 (58.2%) isolates were resistant to two or more drugs. Vancomycin resistant E. faecium (VRE) was not isolated. The transferability of drug-resistance to an E. faecium strain was examined by broth or filter mating. Six (12.5%) of the 48 gentamicin resistance traits, and fifty (50%) of the 101 erythromycin resistance traits were transferred by filter mating. The gentamicin resistance traits of five isolates and the erythromycin resistance traits of four isolates were transferred to the recipient strains by both broth mating and filter mating at a frequency of about 10(-6) and 10(-5) per donor cell, respectively. The five gentamicin resistant strains were shown to harbor pMG1-like plasmids on the basis of their Southern hybridization with pMG1 (65.1 kbp, Gm(r)), which transfers efficiently between enterococci by broth mating. Each of the four erythromycin resistant transconjugants obtained by broth mating harbored a large conjugative plasmid (more than 100 kbp). The plasmids showed no homology with well-characterized enterococcal conjugative plasmids such as pAD1, pPD1, pAM(beta)1, pIP501 and pMG1 by Southern hybridization. Of the erythromycin resistance traits that transferred only by filter mating, it was found that the erythromycin resistance trait was conferred by a 47-kbp transposable element that transferred from the chromosome of the donor strain to different sites within the pheromone responsive plasmid pAD1 (60 kbp) of the recipient strain, suggesting that the erythromycin resistance trait was encoded on a conjugative transposon, which was named Tn950.     

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.     

Prevalence of tet gene and complete genome sequencing of tet gene-encoded plasmid (pAHH01) isolated from Aeromonas species in South Korea

Aims: To investigate the tetracycline resistance related to tet genes in Aeromonas isolates collected from water and diseased fish in South Korea. Methods and Results: A total of 34 Aeromonas strains were examined for their susceptibility to tetracycline using the minimum inhibitory concentration (MIC) assay, and the genetic determinants (tetA to E) were analysed. Among these strains, the tetA and tetE genes were predominant (tetA was found in six strains, and tetE was found in nine strains), and 15 strains were tetracycline‐resistant by the MIC assay. Additionally, the 8979‐bp plasmid that contains the tetE gene was fully sequenced. Conclusions: These data may be important with regard to the spread and persistence of tetracycline resistance genes in the bacterial populations that are present in aquaculture systems. Significance and Impact of the Study: Interestingly, no isolate has previously been shown to harbour three tet genes that are mediated by efflux systems, but the tetA, tetD and tetE genes were all isolated from one strain, which had the highest MIC value for tetracycline among the strains analysed in this study. We also investigated the full‐length plasmid that encoded the tetE gene from a tetracycline‐resistant strain.     

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.     

Molecular characterization of antibiotic resistant Escherichia coli strains isolated from tap and spring waters in a coastal region in Turkey

A hundred and seventeen antibiotic-resistant Escherichia coli strains were isolated from public tap and spring waters which were polluted by fecal coliforms. There were no significant differences between two water sources as to the coliform pollution level (p> 0.05). All E. coli isolates were detected to be resistant to one or more antibiotics tested. Nearly 42% of the isolates showed multiresistant phenotype. Three (2.5%) of these isolates contained class 1 integron. Sequencing analysis of variable regions of the class 1 integrons showed two gene cassette arrays, dfr1-aadA1 and dhfrA17-aadA5. Resistance to ampicillin, tetracycline or trimethoprim-sulfamethoxazole was transferable according to the results of conjugation experiments. The rate of tetracycline resistance was 15%. tet(A)-mediated tetracycline resistance was widespread among tetracycline-resistant E. coli isolates. Genotyping by BOX-polymerase chain reaction (BOX-PCR) showed that some of the strains were epidemiologically related. This is the first report on the prevalence and characterization of class 1 integron-containing E. coli isolates of environmental origin in Turkey.     

Transfer and expression of the tetracyclin resistance transposon Tn925 in Acetobacterium woodii

The Enterococcus faecalis conjugative plasmid pCF10 was used to introduce Tn925 into Acetobacterium woodii by filter mating. Tetracycline resistance was transferred at frequencies of about 10(-6) per donor, but no plasmid DNA was found in the transconjugants. DNA hybridization analyses of HindIII-digested chromosomal DNA demonstrated the insertion of Tn925 at a variety of locations, whereas wild type DNA showed no hybridization at all. The transconjugants were used as donor in mating experiments with tetracycline-sensitive Bacillus subtilis. Transfer of tetracycline resistance was observed at frequencies of 10(-8) per recipient.     

In vitro evaluation of the probiotic properties of human intestinal Bifidobacterium species and selection of new probiotic candidates

Aims: The aim of this work was to identify and select new probiotic strains among majority intestinal bifidobacterial species from healthy Spaniards. Methods and Results: One hundred and eighty isolates belonging to seven Bifidobacterium species were subjected to a subtractive system of in vitro analysis addressing beneficial and undesirable traits. Approx. 45% of the isolates were able to grow in 2% bovine bile, and about 20% of these grew at pH 3·5. Undesirable enzymatic activities, such as α‐chymotrypsin, β‐glucuronidase and N‐acetyl‐β‐glucosaminidase were not detected. Atypical antibiotic resistances were not observed, except for tetracycline resistance in a single strain. Intestinal pathogens were inhibited to some extent by all analysed strains. All selected strains adhered to human epithelial cells in a strain‐dependent manner, and none was able to degrade pig mucin. Conclusions: Based on these in vitro analyses, strains of Bifidobacterium catenulatum, Bifidobacterium longum and Bifidobacterium pseudocatenulatum are here proposed as new probiotic candidates. Significance and Impact of the Study: Although in vivo analyses are still needed, these strains belonging to unusual species in the portfolio of probiotic suppliers are thought to be more appropriated than those currently in use, as they show desirable properties and are preponderant among human intestinal populations.     

Relationship between Pigment Producibility and Drug Resistance in Serratia marcescens

Among the clinical isolates of Serratia marcescens, non-pigmented cells appeared more frequently from pigmented, drug-resistant strains than from pigmented, drug-sensitive strains. Transfer of R plasmid from Escherichia coli to pigmented strains caused spontaneous loss of pigment producibility, whereas such spontaneous loss never occurred in fresh cultures of drug-sensitive strains. The non-pigmented strain was a better recipient of R plasmid from E. coli than was the pigmented strain. R plasmid was transferred from the non-pigmented strain to the pigmented strain at a higher frequency than from E. coli to the pigmented strain. The results of the present investigation suggest that transfer of R plasmid may be one of the reasons for the significant increase of non-pigmented, drug-resistant strains of S. marcescens in nature.     

Phenotypic and genetic characterization of multidrug-resistant <Emphasis Type="Italic">Salmonella</Emphasis> Infantis strains isolated from broiler chicken meats in Turkey

Twenty Salmonella Infantis strains resistant against kanamycin, tetracycline, neomycin, spectinomycin, sulphonamide, nalidixic acid and trimethoprim were selected for this study out of 103 Salmonella strains isolated from broiler samples collected from several markets in the Bolu and Ankara regions of Turkey. The resistance genes aadA1, aphA1, sul1, tet(A), dfrA5/dfrA14 and gyrA were determined for these multidrug-resistant S. Infantis strains. S. Infantis strains contained a mega plasmid with the molecular size of 206 kb. The strains were divided into three groups according to the pulsed field gel electrophoresis patterns of XbaI digested chromosomal DNA. A Ser83→Tyr83 point mutation was found in the gyrA gene of all quinolone-resistant isolates. Filter mating experiments showed that 206 kb plasmid transferred nalidixic acid resistance associated with class I integrons.