Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: identity of laboratory-constructed plasmids and clinical isolates.

The structural gene for ampicillin resistance resides upon a 3.2 X 10(6)-dalton sequence of deoxyribonucleic acid, TnA that can be transposed from replicon to replicon in laboratory experiments. TnA was transposed from a large conjugative plasmid to a small nonconjugative plasmid, RSF1010. Several RSF1010::TnA plasmids isolated in these laboratory experiments have been shown to be identical to plasmids found in clinical isolates. These data provide direct support to the theory that transposition of drug resistance genes play a key role in the evolution of R plasmids.     

Isolation of plasmid deoxyribonucleic acid from Pseudomonas putida.

Conditions suitable for reproducible recovery of covalently closed circular deoxyribonucleic acid from strains of Pseudomonas putida containing degradative plasmids (CAM, SAL, OCT, etc.) have been defined. These degradative plasmids could not be isolated by the usual procedure, whereas RP1, an R factor of the P group, present in the isogenic strain of P. putida, was isolated equally well by either the usual procedure or the modified procedure. Characterization by electron microscopy of RP1 deoxyribonucleic acid confirmed the molecular weight (about 40 X 10(6)) previously determined by sucrose gradient centrifugation.     

Transposition of a plasmid deoxyribonucleic acid sequence that mediates ampicillin resistance: independence from host rec functions and orientation of insertion.

Insertion of the transposable deoxyribonucleic acid sequence that specifies the TEM beta-lactamase (TnA) occurred in at least 19 sites on the 5.5 x 10(6)-dalton plasmid RSF1010. There was no significant difference in the frequency of transposition or in the distribution of TnA insertion sites for recombinant plasmids isolated from recombination-proficient (rec+) or recombination-deficient (rec-) bacterial host cells. The site and orientation of TnA insertions were determined by both heteroduplex analysis and enzymatic digestion with restriction endonucleases. Insertion in the gene encoding for sulfonamide resistance occurred without circular permutation in one or the other of two distinct orientations. Insertions in orientation P were strongly polar on distal gene expression, whereas insertions in orientation M were mutagenic but not polar. In addition, we have observed that TnA elements from different R plasmids show fine structural heterogeneity, and that TnA insertion at a site adjacent to the origin of replication causes an increase in plasmid copy number.     

Involvement of plasmid deoxyribonucleic acid in indoleacetic acid synthesis in Pseudomonas savastanoi.

Olive (or oleander) knot is a plant disease incited by Pseudomonas savastanoi. Disease symptoms consist of tumorous outgrowths induced in the plant by bacterial production of indole-3-acetic acid (IAA). Synthesis of IAA occurs by the following reactions: L-tryptophan leads to indoleacetamide leads to indoleacetic acid, catalyzed by tryptophan 2-monooxygenase and indoleacetamide hydrolase, respectively. Whereas the enzymology of IAA synthesis is well characterized, nothing is known about the genetics of the system. We devised a positive selection for the presence of tryptophan 2-monooxygenase based on its capacity to use as a substrate the toxic tryptophan analogue 5-methyltryptophan. Efficient curing of the bacterium of tryptophan 2-monoxygenase, indoleacetamide hydrolase, and IAA production was obtained by acridine orange treatment. Further, loss of capacity to produce IAA by curing was correlated with loss of a plasmid of 34 X 10(6) molecular weight. This plasmid, here called pIAA1, when reintroduced into Iaa- mutants by transformation, restored tryptophan 2-monooxygenase and indoleacetamide hydrolase activities and production of IAA.     

Nucleotide sequence of Pseudomonas aeruginosa conjugative plasmid pUM505 containing virulence and heavy-metal resistance genes

We determined the complete nucleotide sequence of conjugative plasmid pUM505 isolated from a clinical strain of Pseudomonas aeruginosa. The plasmid had a length of 123,322 bp and contained 138 complete coding regions, including 46% open reading frames encoding hypothetical proteins. pUM505 can be considered a hybrid plasmid because it presents two well-defined regions. The first region corresponded to a larger DNA segment with homology to a pathogenicity island from virulent Pseudomonas strains; this island in pUM505 was comprised of genes probably involved in virulence and genes encoding proteins implicated in replication, maintenance and plasmid transfer. Sequence analysis identified pil genes encoding a type IV secretion system, establishing pUM505 as a member of the family of IncI1 plasmids. Plasmid pUM505 also contained virB4/virD4 homologues, which are linked to virulence in other plasmids. The second region, smaller in length, contains inorganic mercury and chromate resistance gene clusters both flanked by putative mobile elements. Although no genes for antibiotic resistance were identified, when pUM505 was transferred to a recipient strain of P. aeruginosa it conferred resistance to the fluoroquinolone ciprofloxacin. pUM505 also conferred resistance to the superoxide radical generator paraquat. pUM505 could provide Pseudomonas strains with a wide variety of adaptive traits such as virulence, heavy-metal and antibiotic resistance and oxidative stress tolerance which can be selective factors for the distribution and prevalence of this plasmid in diverse environments, including hospitals and heavy metal contaminated soils.     

Cloning, nucleotide sequence, and expression of the chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505.

The chromate resistance determinant of Pseudomonas aeruginosa plasmid pUM505 was cloned into broad-host-range vector pSUP104. The hybrid plasmid containing an 11.1-kilobase insert conferred chromate resistance and reduced uptake of chromate in P. aeruginosa PAO1. Resistance to chromate was not expressed in Escherichia coli. Contiguous 1.6- and 6.3-kilobase HindIII fragments from this plasmid hybridized to pUM505 but not to P. aeruginosa chromosomal DNA and only weakly to chromate resistance plasmids pLHB1 and pMG6. Further subcloning produced a plasmid with an insert of 2,145 base pairs, which was sequenced. Analysis of deletions revealed that a single open reading frame was sufficient to determine chromate resistance. This open reading frame encodes a highly hydrophobic polypeptide, ChrA, of 416 amino acid residues that appeared to be expressed in E. coli under control of the T7 promoter. No significant homology was found between ChrA and proteins in the amino acid sequence libraries, but 29% amino acid identity was found with the ChrA amino acid sequence for another chromate resistance determinant sequenced in this laboratory from an Alcaligenes eutrophus plasmid (A. Nies, D. Nies, and S. Silver, submitted for publication).     

铜绿假单胞菌医院感染调查及耐药分析

目的探讨铜绿假单胞菌(PA)医院感染的临床分布及耐药性,为临床合理使用抗菌药物及控制医院感染提供依据。方法 PA的培养与鉴定严格按照《全国临床检验操作规程》进行。药敏试验采用纸片扩散法(K-B法)。结果收集的359株PA中,分离率居前3位的临床科室依次为ICU、呼吸内科和神经内科,分别占38.1%、17.5%和13.9%;临床标本中以呼吸道标本分离率最高,占62.7%;PA对头孢哌酮/舒巴坦的耐药率最低(9.7%),其次为阿米卡星(13.1%)和头孢他啶(17.8%),另12种抗菌药物的耐药率均较高。结论 PA耐药性已十分严重,临床应根据药敏结果合理使用抗菌药物并加强对细菌耐药性的全面监测。     

Site-specific pegylation of an antimicrobial peptide increases resistance to Pseudomonas aeruginosa elastase

M33 is a branched peptide currently under preclinical characterization for the development of a new antibacterial drug against gram-negative bacteria. Here, we report its pegylation at the C-terminus of the three-lysine-branching core and the resulting increase in stability to Pseudomonas aeruginosa elastase. This protease is a virulence factor that acts by destroying peptides of the native immune system. Peptide resistance to this protease is an important feature for M33-Peg activity against Pseudomonas.     

Apparent fusion of the TOL plasmid with the R91 drug resistance plasmid in Pseudomonas aeruginosa.

The TOL catabolic plasmid was shown to be compatible with the R91 drug resistance plasmid. However, the TOL plasmid was extremely unstable in mutant PA03 of P. aeruginosa. By selecting for stabilization of the TOL plasmid in PA03 harbouring R91, it was possible to isolate a strain in which markers from both R91 and TOL appeared to exist in a single recombinant plasmid. This plasmid, pND3, encoded resistance to carbenicillin, was able to transfer at the same frequency as the R91 plasmid and encoded the ability to grow on m-toluate, p-toluate, m-xylene, p-xylene and toluene. In addition, it was shown to be incompatible with the NAH catabolic plasmid and it could be transferred by transduction. The TOL plasmid could stabilize in PA03 harbouring R91 without fusion with R91, and could stabilize in PA03 in the absence of R91. PA03 harbouring either the recombinant plasmid or the stable TOL plasmid in the absence of R91 could promote bacterial chromosome transfer between mutant derivatives of P. aeruginosa strain PA0.     

Multidrug efflux pumps and antimicrobial resistance in Pseudomonas aeruginosa and related organisms

Pseudomonas aeruginosa is an opportunistic human pathogen characterized by an innate resistance to multiple antimicrobial agents. A major contribution to this intrinsic multidrug resistance is provided by a number of broadly-specific multidrug efflux systems, including MexAB-OprM and MexXY-OprM. In addition, these and two additional tripartite efflux systems, MexCD-OprJ and MexEF-OprN, promote acquired multidrug resistance as a result of mutational hyperexpression of the efflux genes. In addition to antibiotics, these pumps promote export of numerous dyes, detergents, inhibitors, disinfectants, organic solvents and homoserine lactones involved in quorum sensing. The efflux pump proteins are highly homologous and consist of a cytoplasmic membrane-associated drug-proton antiporter of the Resistance-Nodulation-Division (RND) family, an outer membrane channel-forming protein [sometimes called outer membrane factor (OMF)] and a periplasmic membrane fusion protein (MFP). Homologues of these systems have been described in Stenotrophomonas maltophilia, Burkholderia cepacia, Burkholderia pseudomallei and the non-pathogen Pseudomonas putida, where they play a role in export of and resistance to multiple antimicrobial agents and/or organic solvents. Although the natural function of these multidrug efflux systems is largely unknown, their contribution to antibiotic resistance and their conservation in a number of important human pathogens makes them logical targets for therapeutic intervention.     

Homology between the invertible deoxyribonucleic acid sequence that controls flagellar-phase variation in Salmonella sp. and deoxyribonucleic acid sequences in other organisms.

The invertible deoxyribonucleic acid (DNA) segment cloned from Salmonella sp. was radioactively labeled and used as a probe to search for homologous sequences by Southern hybridization. Only one copy of the invertible segment could be found on the Salmonella sp. genome. Partial sequence homology with the invertible region was detected in bacteriophage Mu and P1 DNA by low-stringency hybridization. Under these conditions, no homology was detected with Escherichia coli DNA. A strain of Salmonella sp. defective in phase variation carrying the vH2- allele was also analyzed by DNA-DNA hybridization. The results show that there is sequence divergence between diphasic and vH2- strains within the invertible sequence.     

Classification of OprD sequence and correlation with antimicrobial activity of carbapenem agents in Pseudomonas aeruginosa clinical isolates collected in Japan

A total of 99 clinical isolates of metallo-ß-lactamase-negative Pseudomonas aeruginosa collected in Japan between 1998 and 2001 were studied for their susceptibilities to carbapenem agents and corresponding oprD gene mutations. The OprD sequence of each strain was grouped into two major classes, based on the pattern of alterations. Eighty strains (80.8%) were so-called 'full length type', whose OprD proteins were fully encoded. The remaining 19 strains (19.2%) were so-called 'defective type', which possessed deletions or major alterations that might cause conformational changes in the OprD porin protein. The changes in 'defective type' strains led to 15-, 17- and 23-fold increases in the geometric mean MIC for imipenem, meropenem and biapenem compared with 'full length type' strains, respectively. 'Full length type' strains were further classified into six carbapenem susceptible types with the exception of four carbapenem-resistant subtypes with additional amino acid substitutions at D43, G183, R154, G314, G316. However, 'defective type' strains were classified into four types as follows: 10 strains which contained a stop codon within the coding region; six strains which contained IS; one strain with a short deletion near the C-terminal domain; and two strains without a stop codon in the sequenced region. Western blot analysis using OprD antibody showed that binding abilities of OprD proteins against 'full length type' strains were normal, whereas those against 'defective type' strains were lost without exception. These results indicate that OprD structure and antimicrobial activities for carbapenem agents proved to be highly correlated in P. aeruginosa     

N-Terminal amino acid sequence of pilin isolated from Pseudomonas aeruginosa.

The amino-terminal amino acid sequence of the pili protein from Pseudomonas aeruginosa K pili is presented. The sequence is compared with those reported by others for pilin obtained from Neisseria gonorrhoeae and Moraxella nonlique-faciens. All three sequences are highly homologous, contain only two hydrophilic residues in the first 22 positions, and contain an unusual amino acid, N-monomethylphenylalanine, at the amino terminus.     

Bacterial mutation affecting plasmid maintenance in Pseudomonas aeruginosa.

A bacterial mutation, risA, in Pseudomonas aeruginosa caused growth inhibition at 43 degrees C of risA strains containing P2 plasmids. Incubation at 43 degrees C resulted in selection for clones that had lost P2 plasmids.     

Chromate resistance plasmid in Pseudomonas fluorescens.

Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified. Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance. Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re-acquisition of the chromate resistance phenotype and the plasmid. When pLHB1 was transferred by conjugation to Escherichia coli, the plasmid still conferred chromate resistance.     

Conjugation transfer of plasmid resistance to gentamycin and other antibiotics in clinical strains of Ps. aeruginosa]

A possibility of conjugation transfer of the markers of the plasmid resistance to gentamicin and other antibiotics from 10 clinical strains of Ps. aeruginosa, isolated from burn patients to the recipient strain of Ps. aeruginosa PTO 629 Rfr was shown. The marker of gentamicin resistance was transferred to 100 out of 110 of the exconjugants, i.e. 86.2 per cent. The rate of the conjugation transfer in the crosses between the clincal strains of Ps. aeruginosa and the recipient strain PTO 629 Rfr with respect to the gentamicin marker was about 10--7. The plasmid resistance markers in the clincal strains Ps. aeruginosa were transferred in various combinations. Transfer of the markers of resistance to streptomycin, carbenicillin, neomycin and combinations Sm, Nm and Sm, Nm, Cm was not achieved.