喹诺酮外排泵耐药基因oqxAB的耐药机制及其流行情况

oqx AB是编码Oqx AB外排泵蛋白的多重耐药基因,由2个开放阅读框oqx A和oqx B组成,其主要介导细菌对喹诺酮类药物的耐药。该基因最早是在大肠埃希菌pOLA52质粒上被发现,是质粒介导的喹诺酮耐药基因的重要成员之一。目前已经分别发现了11个oqxA和32个oqxB等位基因。oqxAB通过质粒进行水平传播,使受体菌株容易形成耐药性,增加了临床治疗的难度。该基因目前主要流行于肠杆菌科,但不能排除也存在于非肠杆菌科细菌,给人类与家畜健康造成巨大的威胁。本文综述了喹诺酮外排泵耐药基因oqxAB及其等位基因的发现、耐药机制、国内外流行特点,以期为临床和畜牧生产中合理使用喹诺酮类抗菌药物,减少oqxAB基因的流行提供资料。     

Identification of regions of the Streptococcus faecalis plasmid pCF-10 that encode antibiotic resistance and pheromone response functions

The conjugative plasmid pCF-10 (58 kb) of Streptococcus faecalis has been mapped with restriction enzymes. By restriction mapping and Southern hybridization analysis, a 16-kb segment of the plasmid was shown to resemble closely the conjugative tetracycline resistance transposon, Tn916. Mutagenesis of the plasmid with the erythromycin resistance transposon Tn917 was used to localize a tetracycline resistance determinant and several regions involved in conjugal transfer. Fifty Tn917 insertions (outside the region of the plasmid homologous to Tn916) affecting mating behavior and the ability of donor cells to respond to the sex pheromone cCF-10 were mapped to nine distinct segments, or tra regions. Insertions into tra regions 1-3 and 7-9 led to an enhanced transfer ability of mutant plasmids relative to the transfer frequency obtained for the wild-type plasmid. Cells carrying these mutant plasmids differed in colony morphology or growth in broth culture from cells carrying pCF-10. Insertions into tra regions 4-6 resulted in reduced plasmid transfer, or completely eliminated the mating potential of donor cells. Insertions generating transfer-defective plasmids could be grouped further according to the ability of strains harboring the mutant plasmids to respond to cCF-10. HindIII fragments of pCF-10 coding for transfer functions have been cloned into Escherichia coli.     

Analysis of pSC138, the multidrug resistance plasmid of Salmonella enterica serotype Choleraesuis SC-B67

Salmonella enterica serotype Choleraesuis (S. Choleraesuis) usually causes systemic infections in man and needs antimicrobial treatment. Multidrug resistance (MDR) in S. Choleraesuis is thus a great concern in the treatment of systemic non-typhoid salmonellosis. A large plasmid, pSC138, was identified in 2002 from a S. Choleraesuis strain SC-B67 that was resistant to all antimicrobial agents commonly used to treat salmonellosis, including ciprofloxacin and ceftriaxone. Complete DNA sequence of the plasmid had been determined previously (Chiu et al., 2005). In the present study, the sequence of pSC138 was reannotated in detail and compared with several newly sequenced plasmids. Some transposable elements and drug resistance genes were further delineated. Plasmid pSC138 was 138,742 bp in length and consisted of 177 open reading frames (ORFs). While 134 of the ORFs displayed significant identity levels to other plasmid and prokaryotic sequences, the remaining 43 ORFs have not been previously reported. Mobile elements, including two integrons, seven insertion sequences and eight transposons, and a truncated prophage together encompass at least 66,781 bp (48.1%) of the plasmid genome. The sequence of pSC138 consists of three major regions: a large composite transposable region Tn6088 with a Tn21-like backbone inserted by a variety of integrons or transposable elements; a transfer/maintenance region that contains a conserved ISEcp1-mediated transposon-like element Tn6092, carrying an AmpC gene, bla(CMY-2), that confers the ceftriaxone resistance; and a Rep_3 type of replication region. Another seven bacteremic strains of S. Choleraesuis that expressed the same MDR phenotype were identified during 2003-2008. The same Rep_3 type replicase and the bla(CMY-2)-containing, ISEcp1-mediated transposon-like element were found in the MDR isolates, suggesting a successful preservation and dissemination of the MDR plasmid. Comparison of pSC138 with other recently published plasmids revealed a high identity level between partial sequences of pSC138 and plasmids of the same or different incompatibility groups. The large MDR region found in pSC138 may provide a niche for the future evolution of the plasmid by acquisition of relevant resistance genes through the panoply of mobile elements and illegitimate recombination events.     

Expansion of the IncX plasmid family for improved identification and typing of novel plasmids in drug-resistant Enterobacteriaceae

IncX plasmids are narrow host range plasmids of Enterobactericeae that have been isolated for over 50years. They are known to encode type IV fimbriae enabling their own conjugative transfer, and to provide accessory functions to their host bacteria such as resistance towards antimicrobial agents and biofilm formation. Previous plasmid-based replicon typing procedures have indicated that the prevalence of IncX plasmids is low among members of the Enterobacteriaceae. However, examination of a number of IncX-like plasmid sequences and their occurrence in various organisms suggests that IncX plasmid diversity and prevalence is underappreciated. To address these possible shortcomings, we generated additional plasmid sequences of IncX plasmids of interest and compared them to the genomes of all sequenced IncX-like plasmids. These comparisons revealed that IncX plasmids possess a highly syntenic plasmid backbone, but that they are quite divergent with respect to nucleotide and amino acid similarity. Based on phylogenetic comparisons of the sequenced IncX plasmids, the IncX plasmid group has been expanded to include at least four subtypes, IncX1-IncX4. A revised IncX plasmid replicon typing procedure, based upon these sequences and subtypes, was then developed. Use of this revised typing procedure revealed that IncX plasmid occurrence among bacterial populations is much more common than had previously been acknowledged. Thus, this revised procedure can be used to better discern the occurrence of IncX type plasmids among enterobacterial populations.     

Emergence of a globally dominant IncHI1 plasmid type associated with multiple drug resistant typhoid

Typhoid fever, caused by Salmonella enterica serovar Typhi (S. Typhi), remains a serious global health concern. Since their emergence in the mid-1970s multi-drug resistant (MDR) S. Typhi now dominate drug sensitive equivalents in many regions. MDR in S. Typhi is almost exclusively conferred by self-transmissible IncHI1 plasmids carrying a suite of antimicrobial resistance genes. We identified over 300 single nucleotide polymorphisms (SNPs) within conserved regions of the IncHI1 plasmid, and genotyped both plasmid and chromosomal SNPs in over 450 S. Typhi dating back to 1958. Prior to 1995, a variety of IncHI1 plasmid types were detected in distinct S. Typhi haplotypes. Highly similar plasmids were detected in co-circulating S. Typhi haplotypes, indicative of plasmid transfer. In contrast, from 1995 onwards, 98% of MDR S. Typhi were plasmid sequence type 6 (PST6) and S. Typhi haplotype H58, indicating recent global spread of a dominant MDR clone. To investigate whether PST6 conferred a selective advantage compared to other IncHI1 plasmids, we used a phenotyping array to compare the impact of IncHI1 PST6 and PST1 plasmids in a common S. Typhi host. The PST6 plasmid conferred the ability to grow in high salt medium (4.7% NaCl), which we demonstrate is due to the presence in PST6 of the Tn6062 transposon encoding BetU.     

Sequencing of IncX-Plasmids Suggests Ubiquity of Mobile Forms of a Biofilm-Promoting Gene Cassette Recruited from Klebsiella pneumoniae

Plasmids are a highly effective means with which genetic traits that influence human health, such as virulence and antibiotic resistance, are disseminated through bacterial populations. The IncX-family is a hitherto sparsely populated group of plasmids that are able to thrive within Enterobacteriaceae. In this study, a replicon-centric screening method was used to locate strains from wastewater sludge containing plasmids belonging to the IncX-family. A transposon aided plasmid capture method was then employed to transport IncX-plasmids from their original hosts (and co-hosted plasmids) into a laboratory strain (Escherichia coli Genehogs®) for further study. The nucleotide sequences of the three newly isolated IncX-plasmids (pLN126_33, pMO17_54, pMO440_54) and the hitherto un-sequenced type-plasmid R485 revealed a remarkable occurrence of whole or partial gene cassettes that promote biofilm-formation in Klebsiella pneumonia or E. coli, in all four instances. Two of the plasmids (R485 and pLN126_33) were shown to directly induce biofilm formation in a crystal violet retention assay in E. coli. Sequence comparison revealed that all plasmid-borne forms of the type 3 fimbriae encoding gene cassette mrkABCDF were variations of a composite transposon Tn6011 first described in the E. coli IncX plasmid pOLA52. In conclusion, IncX-plasmids isolated from Enterobacteriaceae over almost 40 years and on three different continents have all been shown to carry a type 3 fimbriae gene cassette mrkABCDF stemming from pathogenic K. pneumoniae. Apart from contributing general knowledge about IncX-plasmids, this study also suggests an apparent ubiquity of a mobile form of an important virulence factor and is an illuminating example of the recruitment, evolution and dissemination of genetic traits through plasmid-mediated horizontal gene transfer.     

Plasmid pB8 is closely related to the prototype IncP-1beta plasmid R751 but transfers poorly to Escherichia coli and carries a new transposon encoding a small multidrug resistance efflux protein

The IncP-1beta plasmid pB8, which confers resistance to amoxicillin, spectinomycin, streptomycin, and sulfonamides, was previously isolated from a sewage treatment plant. It was found to possess abnormal conjugative transfer properties, i.e., transfer to Escherichia coli by conjugation or electroporation could not be detected. We showed in this study that plasmid pB8 is transferable to E. coli by conjugation, but only at low frequencies and under specific experimental conditions, a phenomenon that is very unusual for IncP-1 plasmids. Determination of the complete 57,198bp pB8 nucleotide sequence revealed that the backbone of the plasmid consists of a complete set of IncP-1beta-specific genes for replication initiation, conjugative plasmid transfer, stable inheritance, and plasmid control with an organisation identical to that of the prototype IncP-1beta plasmid R751. All of the minor differences in the pB8 backbone sequence compared to that of R751 were also found in other IncP-1beta plasmids known to transfer to and replicate in E. coli. Plasmids pB8 and R751 can be distinguished with respect to their accessory genetic elements. First, the pB8 region downstream of the replication initiation gene trfA contains two transposable elements one of which is similar to Tn5501. The latter transposon encodes a putative post-segregational-killing system and the small multidrug resistance (SMR) protein QacF, mediating quaternary ammonium compound resistance. The accessory genes in this region are not responsible for the poor plasmid transfer to E. coli since a pB8 deletion derivative devoid of all genes in that region showed the same conjugative transfer properties as pB8. A Tn5090/Tn402 derivative carrying a class 1 integron is located between the conjugative transfer modules. The Tn5090/Tn402 integration-sites are exactly identical on pB8 and R751 but in contrast to R751 the pB8 element carries the resistance gene cassettes oxa-2 for amoxicillin resistance and aadA4 for streptomycin/spectinomycin resistance, the integron-specific conserved segment consisting of the genes qacEDelta1, sul1, and orf5, and a truncated tni transposition module (tniAB). Although future work will have to determine the molecular basis for the poor transfer of pB8 to E. coli, our findings demonstrate that the host-range of typical IncP-1 plasmids may be less broad than expected.     

Characterization of incompatibility group HI1 plasmids from Salmonella typhi by restriction endonuclease digestion and hybridization of DNA probes for Tn3, Tn9, and Tn10

Chloramphenicol resistance in Salmonella typhi is medicated by plasmids of the incompatibility group H, subgroup 1 (IncHI1). Eight IncHI1 plasmids from S. typhi strains originating in Mexico, Vietnam, Thailand, and India were examined by restriction enzyme digestion. The restriction enzymes, Apal, Xbal, and PstI were found to be most useful for comparison of plasmid DNAs. Four plasmids from S. typhi isolated in Mexico, Vietnam, and Thailand between 1972 and 1974 had identical restriction patterns with all three enzymes. The other IncHI1 plasmids showed only minor differences. However, some significant differences were noted between these IncHI1 plasmids and the prototype IncHI1 plasmid R27, which was isolated from S. typhimurium in 1961 and for which a restriction map has been constructed. Southern transfer hybridization with a nick-translated HI1 plasmid as a probe confirmed that there is a great deal of sequence homology among the IncHI1 plasmids. DNA probes were used to locate DNA sequences for ampicillin resistance (Tn3), chloramphenicol resistance (Tn9), tetracycline resistance (Tn10), and the one-way incompatibility between IncHI1 plasmids and the F factor, a characteristic property of IncHI1 plasmids. The results demonstrate that IncHI1 plasmids isolated from S. typhi from widely different geographic sources are very similar. Comparisons between the S. typhi plasmids and R27 indicated that conserved regions of DNA were those involved in conjugative transfer.     

Molecular Characterization of an Atypical IncX3 Plasmid pKPC-NY79 Carrying bla KPC-2 in a Klebsiella pneumoniae

The IncX family of plasmids has recently been expanded to include at least four subtypes, IncX1–IncX4. The revised classification provides an opportunity for improving our understanding of the sequence diversity of the IncX plasmids and the resistance genes they carried. We described the complete nucleotide sequence of a novel IncX3 plasmid, pKPC-NY79 (42,447 bp) from a sequence-type 258 Klebsiella pneumoniae strain that was isolated from a patient who was hospitalized in New York, United States. In pKPC-NY79, the plasmid scaffold and genetic load region were highly similar to homologous regions in pIncX-SHV (IncX3, JN247852) and the bla _KPC carrying pKpQIL (IncFII_k, GU595196), respectively, indicating that it has possibly arisen through recombination of plasmids. The bla _KPC-2 gene, as part of a transposon Tn4401a, was found within the genetic load region. The backbone of pKPC-NY79 differs from pIncX-SHV by a deletion involving the gene tandem hns–topB (encoding H-NS protein and topoisomerase III, respectively) and a putative ATPase gene. Unexpectedly, the impact of the hns–topB deletion on host fitness and plasmid stability was found to be small. In conclusion, the findings contribute to a better understanding of the plasmid platforms carrying bla _KPC and of variations in the backbone of the IncX3 plasmids.     

Mapping of transfer regions within incompatibility group HI plasmid R27.

Plasmids of incompatibility group HI are large (greater than 150 kilobases [kb]) and possess an unusual thermosensitive mode of conjugative transfer. R27, the prototype IncHi1 plasmid, encodes resistance to tetracycline via a determinant which is related to transposon Tn10. A restriction endonuclease map of R27 (size, 182 kb) was recently constructed with ApaI, PstI, and XbaI. Transfer genes within R27 were mapped by insertion of Tn5 and Tn7. At least two different regions of the plasmid were concerned with transfer functions. Insertions into either region completely abolished transfer. None of the insertions had any effect on entry exclusion (Eex) of other IncH plasmids. However, a deletion mutant which lacked the Eex function was obtained, allowing us to map the probable site of the gene encoding Eex to one of the two transfer regions. The tetracycline resistance determinant in R27 was located within an 8-kb region between the two main transfer regions. The transfer genes, therefore, are not located together in R27 but are situated in at least two major widely separated transfer regions.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

Design and synthesis of a quintessential self-transmissible IncX1 plasmid, pX1.0

DNA exchange in bacteria via conjugative plasmids is believed to be among the most important contributing factors to the rapid evolution- and diversification rates observed in bacterial species. The IncX1 plasmids are particularly interesting in relation to enteric bacteria, and typically carry genetic loads like antibiotic resistance genes and virulence factors. So far, however, a "pure" version of these molecular parasites, without genetic loads, has yet to be isolated from the environment. Here we report the construction of pX1.0, a fully synthesized IncX1 plasmid capable of horizontal transfer between different enteric bacteria. The designed pX1.0 sequence was derived from the consensus gene content of five IncX1 plasmids and three other, more divergent, members of the same phylogenetic group. The pX1.0 plasmid was shown to replicate stably in E. coli with a plasmid DNA per total DNA ratio corresponding to approximately 3-9 plasmids per chromosome depending on the growth phase of the host. Through conjugation, pX1.0 was able to self-transfer horizontally into an isogenic strain of E. coli as well as into two additional species belonging to the family Enterobacteriaceae. Our results demonstrate the immediate applicability of recent advances made within the field of synthetic biology for designing and constructing DNA systems, previously existing only in silica.     

A facile and reversible method to decrease the copy number of the ColE1-related cloning vectors commonly used in Escherichia coli.

We report a technique which uses the cointegrate intermediate of transposon Tn1000 transposition as a means to lower the copy number of ColE1-type plasmids. The transposition of Tn1000 from one replicon to another is considered a two-step process. In the first step, the transposon-encoded TnpA protein mediates fusion of the two replicons to produce a cointegrate. In the second step, the cointegrate is resolved by site-specific recombination between the two transposon copies to yield the final transposition products: the target replicon with an integrated transposon plus the regenerated donor replicon. Using in vitro techniques, the DNA sequence of the Tn1000 transposon was altered so that cointegrate formation occurs but resolution by the site-specific recombination pathway is blocked. When this transposon was resident on an F factor-derived plasmid, a cointegrate was formed between a multicopy ColE1-type target plasmid and the conjugative F plasmid. Conjugational transfer of this cointegrate into a polA strain resulted in a stable cointegrate in which replication from the ColE1 plasmid origin was inhibited and replication proceeded only from the single-copy F factor replication origin. We assayed isogenic strains which harbored plasmids encoding chloramphenicol acetyltransferase to measure the copy number of such F factor-ColE1-type cointegrate plasmids and found that the copy number was decreased to the level of single-copy chromosomal elements. This method was used to study the effect of copy number on the expression of the fabA gene (which encodes the key fatty acid-biosynthetic enzyme beta-hydroxydecanoylthioester dehydrase) by the regulatory protein encoded by the fadR gene.     

The complete nucleotide sequence of the resistance plasmid R478: defining the backbone components of incompatibility group H conjugative plasmids through comparative genomics

Horizontal transfer of resistance determinants amongst bacteria can be achieved by conjugative plasmid DNA elements. We have determined the complete 274,762 bp sequence of the incompatibility group H (IncH) plasmid R478, originally isolated from the Gram negative opportunistic pathogen Serratia marcescens. This self-transferable extrachromosomal genetic element contains 295 predicted genes, of which 144 are highly similar to coding sequences of IncH plasmids R27 and pHCM1. The regions of similarity among these three IncH plasmids principally encode core plasmid determinants (i.e., replication, partitioning and stability, and conjugative transfer) and we conducted a comparative analysis to define the minimal IncHI plasmid backbone determinants. No resistance determinants are included in the backbone and most of the sequences unique to R478 were contained in a large contiguous region between the two transfer regions. These findings indicate that plasmid evolution occurs through gene acquisition/loss predominantly in regions outside of the core determinants. Furthermore, a modular evolution for R478 was signified by the presence of gene neighbors or operons that were highly related to sequences from a wide range of chromosomal, transposon, and plasmid elements. The conjugative transfer regions are most similar to sequences encoded on SXT, Rts1, pCAR1, R391, and pRS241d. The dual partitioning modules encoded on R478 resemble numerous sequences; including pMT1, pCTX-M3, pCP301, P1, P7, and pB171. R478 also codes for resistance to tetracycline (Tn10), chloramphenicol (cat), kanamycin (aphA), mercury (similar to Tn21), silver (similar to pMG101), copper (similar to pRJ1004), arsenic (similar to pYV), and tellurite (two separate regions similar to IncHI2 ter determinants and IncP kla determinants). Other R478-encoded sequences are related to Tn7, IS26, tus, mucAB, and hok, where the latter is surrounded by insLKJ, and could potentially be involved in post-segregation killing. The similarity to a diverse set of bacterial sequences highlights the ability of horizontally transferable DNA elements to acquire and disseminate genetic traits through the bacterial gene pool.     

Characterisation of plasmids purified from Acetobacter pasteurianus 2374

Four cryptic plasmids pAP1, pAP2, pAP3, and pAP4 with their replication regions AP were isolated from Gram-negative bacteria Acetobacter pasteurianus 2374 and characterised by sequence analyses. All plasmids were carrying the kanamycin resistance gene. Three of four plasmids pAP2, pAP3, and pAP4 encode an enzyme that confers ampicillin resistance to host cells. Moreover, the tetracycline resistance gene was identified only in pAP2 plasmid. All plasmids are capable to coexist with each other in Acetobacter cells. On the other hand, the coexistence of more than one plasmid is excluded in Escherichia coli. The nucleotide sequence of replication regions showed significant homology. The nucleotide and protein sequence analyses of resistance genes of all plasmids were compared with transposons Tn3, Tn10, and Tn903 which revealed significant differences in the primary structure, however no functional changes of gene were obtained.     

Comparative genomics of multidrug resistance-encoding IncA/C plasmids from commensal and pathogenic Escherichia coli from multiple animal sources

Incompatibility group A/C (IncA/C) plasmids have received recent attention for their broad host range and ability to confer resistance to multiple antimicrobial agents. Due to the potential spread of multidrug resistance (MDR) phenotypes from foodborne pathogens to human pathogens, the dissemination of these plasmids represents a public health risk. In this study, four animal-source IncA/C plasmids isolated from Escherichia coli were sequenced and analyzed, including isolates from commercial dairy cows, pigs and turkeys in the U.S. and Chile. These plasmids were initially selected because they either contained the floR and tetA genes encoding for florfenicol and tetracycline resistance, respectively, and/or the bla(CMY-2) gene encoding for extended spectrum β-lactamase resistance. Overall, sequence analysis revealed that each of the four plasmids retained a remarkably stable and conserved backbone sequence, with differences observed primarily within their accessory regions, which presumably have evolved via horizontal gene transfer events involving multiple modules. Comparison of these plasmids with other available IncA/C plasmid sequences further defined the core and accessory elements of these plasmids in E. coli and Salmonella. Our results suggest that the bla(CMY-2) plasmid lineage appears to have derived from an ancestral IncA/C plasmid type harboring floR-tetAR-strAB and Tn21-like accessory modules. Evidence is mounting that IncA/C plasmids are widespread among enteric bacteria of production animals and these emergent plasmids have flexibility in their acquisition of MDR-encoding modules, necessitating further study to understand the evolutionary mechanisms involved in their dissemination and stability in bacterial populations.     

Transfer-defective and tetracycline-sensitive mutants of the incompatibility group HI plasmid R27 generated by insertion of transposon 7

Transposon Tn7 insertion was used to obtain either transfer-defective (Tra-) or tetracycline-sensitive (Tc-) mutants of the HI incompatibility group (IncHI) plasmid R27. The 600 apparent R27::Tn7 derivatives fell into three classes: Tra-, Tc-, and Tra- Tc-. Mutants of R27 defective in the thermosensitive mode of transfer characteristic of IncH plasmids were obtained with transfer frequencies of less than 1 X 10(-8) transconjugants per recipient after 18 hr at 26 degrees C. These mutants, which were generated at a frequency of 1 per 100 insertions, were nonleaky and nonrevertible. Tc- mutants of R27, generated at a frequency of 0.5 per 100 insertions, were also nonrevertible. Loss of tetracycline resistance was associated with an increased frequency of transfer (average 3.6 X 10(-3) transconjugants per donor per hour at 30 degrees C) compared with transfer of the wild-type R27 plasmid (1.6 X 10(-8) per donor per hour). Tn7 insertions which generated Tc- or Tra- mutants of R27 had no effect on entry exclusion of other H group plasmids. The molecular weights of Tra- and Tc- R27::Tn7 derivatives were approximately 120.5 MDa, corresponding to the sum of R27 (112 MDa) and Tn7 (8.5 MDa). A third class of Tn7 insertion derivatives (Tra- Tc-) was obtained; however, strains expressing this phenotype were plasmid free, and appeared to have Tn7 integrated at a chromosomal site. Restriction digestion with XbaI and subsequent hybridization with ColE1::Tn7 were used to compare R27::Tn7 derivatives and to locate Tn7 insertion sites. Loss of tetracycline resistance was associated with Tn7 insertion into a 24-kb XbaI fragment of R27. Although loss of plasmid transfer in several R27::Tn7 derivatives was accompanied by insertion of Tn7 into a 14-kb XbaI fragment of the plasmid, these mutants had also undergone a small increase in the size of the 24-kb XbaI fragment of R27.     

Dissemination of the Transmissible Quinolone-Resistance Gene qnrS1 by IncX Plasmids in Nigeria

The plasmid-encoded quinolone resistance gene qnrS1 was recently found to be commonly associated with ciprofloxacin resistance in Nigeria. We mapped the qnrS1 gene from an Escherichia coli isolate obtained in Nigeria to a 43.5 Kb IncX2 plasmid. The plasmid, pEBG1, was sufficient to confer ciprofloxacin non-susceptibility, as well as tetracycline and trimethoprim resistance, on E. coli K-12. Deletion analysis confirmed that qnrS1 accounted for all the ciprofloxacin non-suceptibility conferred by pEBG1 and tetracycline and trimethoprim resistance could be attributed to tetAR and dfrA14 genes respectively. While it contained a complete IncX conjugation system, pEBG1 was not self-transmissible likely due to an IS3 element inserted between the pilX5 and pilX6 genes. The plasmid was however efficiently mobilizable. pEBG1 was most similar to another qnrS1-bearing IncX2 plasmid from Nigeria, but both plasmids acquired qnrS1 independently and differ in their content of other resistance genes. Screening qnrS1–positive isolates from other individuals in Nigeria revealed that they carried neither pEBG1 nor pNGX2-QnrS1 but that IncX plasmids were prevalent. This study demonstrates that the IncX backbone is a flexible platform that has contributed to qnrS1 dissemination in Nigeria.     

Transposition of Tn904 encoding streptomycin resistance into the octopine Ti plasmid of Agrobacterium tumefaciens.

A transfer-deficient derivative of plasmid RP1-pMG1 was isolated after insertion of Mu cts62. The Tra- R plasmid was used to donate Tn904, encoding streptomycin resistance, to Ti plasmid pAL102 harbored by Agrobacterium tumefaciens Ach5. Under conditions promoting high Ti transfer frequencies, 155 strains were isolated in which the streptomycin marker coupled with Ti plasmid in further transfer experiments. These isolates represent stable insertions of Tn904 into the Ti plasmid. In addition, 19 strains were isolated in which the insertion of Tn904 was apparently unstable. The frequency of stable Tn904 transpositions was estimated to be 3 x 10(4-) per transferred Ti plasmid. Evidence was obtained that Tn904 readily may transpose from the Ti plasmid into the bacterial chromosome. The strains carrying Ti plasmids with stable insertions were characterized with respect to virulence, octopine degradation, octopine synthesis in induced tumors, and Ti plasmid transfer. Thirteen of the strains were found to be affected in tumor-inducing ability.