Sequence of the R1 plasmid and comparison to F and R100.

The R1 antibiotic resistance plasmid, originally discovered in a clinical Salmonella isolate in London, 1963, has served for decades as a key model for understanding conjugative plasmids. Despite its scientific importance, a complete sequence of this plasmid has never been reported. We present the complete genome sequence of R1 along with a brief review of the current knowledge concerning its various genetic systems and a comparison to the F and R100 plasmids. R1 is 97,566 nucleotides long and contains 120 genes. The plasmid consists of a backbone largely similar to that of F and R100, a Tn21-like transposon that is nearly identical to that of R100, and a unique 9-kb sequence that bears some resemblance to sequences found in certain Klebsiella oxytoca strains. These three regions of R1 are separated by copies of the insertion sequence IS1. Overall, the structure of R1 and comparison to F and R100 suggest a fairly stable shared conjugative plasmid backbone into which a variety of mobile elements have inserted to form an "accessory" genome, containing multiple antibiotic resistance genes, transposons, remnants of phage genes, and genes whose functions remain unknown.

Plasmid-Encoded AmpC (pAmpC) in Enterobacteriaceae: epidemiology of microorganisms and resistance markers.

CMY-2 Β-lactamase is an important cause of Β-lactam resistance in Enterobacteriaceae and constitutes the most widespread pAmpC. Although CMY-2 has been previously recognized in our region, the real prevalence and epidemiology of this resistance marker was uncertain. During August-October 2009, we conducted a multicenter, prospective study to determine pAmpC prevalence and to characterize CMY-2 producing Escherichia coli associated plasmids. Plasmid-encoded AmpC prevalence was 0.9 % in enterobacteria in this period, being CMY-2 prevalent and to a lesser extent DHA. Molecular typing of CMY-2- producing Escherichia coli isolates showed several lineages. Moreover, replicon typing of cmy-2- containing plasmids displayed a broad diversity in Inc/cmy-2 links. Therefore, association of cmy-2 with specific transposon elements may be responsible for the spread of this resistance marker in Enterobacteriaceae.

Pentapeptide-repeat proteins that act as topoisomerase poison resistance factors have a common dimer interface.

The protein AlbG is a self-resistance factor against albicidin, a nonribosomally encoded hybrid polyketide-peptide with antibiotic and phytotoxic properties produced by Xanthomonas albilineans. Primary-sequence analysis indicates that AlbG is a member of the pentapeptide-repeat family of proteins (PRP). The structure of AlbG from X. albilineans was determined at 2.0 Šresolution by SAD phasing using data collected from a single trimethyllead acetate derivative on a home source. AlbG folds into a right-handed quadrilateral ß-helix composed of approximately eight semi-regular coils. The regularity of the ß-helix is blemished by a large loop/deviation in the ß-helix between coils 4 and 5. The C-terminus of the ß-helix is capped by a dimerization module, yielding a dimer with a 110 Šsemi-collinear ß-helical axis. This method of dimer formation appears to be common to all PRP proteins that confer resistance to topoisomerase poisons and contrasts with most PRP proteins, which are typically monomeric.

First report of plasmid-mediated quinolone resistance qnrA1 gene in Klebsiella pneumoniae isolate of animal origin.

One QnrA1-producing Klebsiella pneumoniae isolate GDKA1 from chicken was detected. The qnrA1 gene on plasmid pGDKA1 was located in a genetic environment similar to that in In36 on plasmid pHSH1 and could be cotransferred to Escherichia coli J53 Az(R) with other resistances by a conjugation experiment. Upstream of the qnrA1 gene, there was a class I integron with the dfrA27 and aadA2 cassettes. Similar genetic environments of qnrA1 in Enterobacteriaceae isolates from both human and animal origin might, to some extent, demonstrate similar mechanisms of qnrA distribution. The presence of qnrA1 in health animal commensal bacteria should be worthy of note. This is the first report of qnrA1 in K. pneumoniae and dfrA27 in an Enterobacteriaceae isolate of animal origin.

Complete genome sequence of the incompatibility group I1 plasmid R64.

A streptomycin and tetracycline resistance plasmid R64 isolated from Salmonella enterica serovar Typhimurium belongs to the incompatibility group I1 (IncI1). The DNA sequence of the R64 conjugative transfer region was described previously (Komano et al., 2000). Here, we report the complete genome sequence of R64. In the circular double-stranded R64 genome with 120,826bp, 126 complete ORFs are predicted. In addition, 2 and 6 different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of five major regions: replication, drug resistance, stability, transfer leading, and conjugative transfer regions in clockwise order. The nucleotide sequence essential for autonomous replication of R64 is completely identical to that of IncI1 colicinogenic plasmid ColIb-P9, an indication that these two plasmids share the same mechanisms for replication and copy number control. Tetracycline and streptomycin resistance genes are encoded in transposons Tn10 and Tn6082, respectively. These transposons and two insertion elements, IS2 and IS1133, were inserted stepwise into the arsenic-resistant gene, arsA1, present in the drug resistance region. The stability and transfer leading regions contain various important genes such as parAB, resD, ardA, psiAB, or ssb for plasmid maintenance, recombination and transfer reactions. When the genome of R64 was compared with those of other plasmids, varying levels of similarity were observed. It is suggested that genetic recombinations including the site-specific rfsF-ResD system have played an important role in diversity of genomes related to R64. It was found that R64 exhibits highly organized genome structure.

[A novel Salmonella Typhimurium plasmid, pAnkS: an example for plasmid evolution in antibiotic resistance]. / Yeni bir Salmonella Typhimurium plazmidi, pAnkS: antibiyotik direnç gelisiminde plazmid evriminin bir örnegi.

In this study, a plasmid, carrying ampicillin resistance (ampR) gene, isolated from a clinical isolate of Salmonella enterica serotype Typhimurium presenting ACSSuT (ampicilin, chloramphenicol, streptomycin, sulphonamide, tetracycline) resistance phenotype, was defined. The length of complete sequence of this plasmid was 8271 base pairs (bp), and it was named as pAnkS owing to its isolation place (plasmid-Ankara- Salmonella). The plasmid was analyzed for potential reading frames and structural features indicative of transposons and transposon relics. The Xmnl enzyme restriction fragments of pAnkS were cloned into E. coli plasmid vectors (pBSK), sequenced and analyzed with the BLAST programs. Plasmid pAnkS has contained a previously defined enterohemorrhagic E. coli (EHEC) plasmid p4821 as a core region and also contained a complete Tn3-like transposon of 4950 bp consisting of the left terminal repeat, Tn3-related tnpR and tnpA genes for transposition functions, ampicillin resistance gene bla(TEM), and the right terminal repeats, pAnkS showed strong homology with another Salmonella plasmid, pNTP16, for sequences that belong to p4821 and partial Tn3 segments. It was found that pNTP16 also carries kanamycin resistance gene (kanR) in addition to ampR gene. Plasmid pAnkS is one of the few completely sequenced plasmids from Salmonella Typhimurium and is in the middle of the pathway of evolution of plasmid from p4821 to pNTP16. The identification of pAnkS might help better understanding of plasmid evolution.

Molecular characterization of antimicrobial resistance in Shigella sonnei isolates in Korea.

The antimicrobial resistance of 122 Shigella sonnei isolates obtained in Korea during the period 1991-2000 was characterized. These isolates were highly resistant to traditional antibiotics such as trimethoprim (100 %), streptomycin (100 %), sulfamethoxazole (94 %), tetracycline (93 %) and nalidixic acid (90 %). All S. sonnei isolates carried Tn7 in their chromosomes. The 8.4 kb non-transferable resistance (R) plasmid carrying tetA, strA-strB and sul1 was found in 93 % of the S. sonnei isolates. Resistance to nalidixic acid first appeared in a S. sonnei isolate in 1997, and then in all S. sonnei isolates from 1998 and 1999. Resistance to commonly prescribed antibiotics such as ampicillin was increased in S. sonnei isolates during the outbreak period 1998-2000. Resistance to ampicillin was mediated by the conjugative R plasmids carrying blaTEM-1. In conclusion, S. sonnei acquired antimicrobial resistance to commonly prescribed antibiotics through the horizontal transfer of conjugative R plasmids, while the genetic stability of transposon and non-transferable R plasmids was responsible for resistance to traditional antibiotics.

Characterization of a novel type of MLSB resistance plasmid from Staphylococcus saprophyticus carrying a constitutively expressed erm(C) gene.

An erm(C)-carrying plasmid of unusual size and restriction map, designated pSES22, was identified in a Staphylococcus saprophyticus strain and sequenced completely. Constitutive expression of the erm(C) gene from pSES22 is based on a novel 22-bp tandem duplication in the erm(C) translational attenuator. Comparative analysis of the deduced Erm(C) amino acid sequence revealed that Erm(C) from pSES22 - together with an Erm(C) methylase from S. hyicus - represented a separate branch in the homology tree of Erm(C) methylases. Structural comparisons showed that plasmid pSES22 differed distinctly from all other completely sequenced erm(C)-carrying resistance plasmids. However, pSES22 was similar to several members of a diverse group of small plasmids, all of which carried closely related plasmid backbones consisting of the genes repU and pre/mob, but differed in their resistance genes.

The complete DNA sequence and analysis of R27, a large IncHI plasmid from Salmonella typhi that is temperature sensitive for transfer.

Salmonella typhi, the causative agent of typhoid fever, annually infects 16 million people and kills 600 000 world wide. Plasmid-encoded multiple drug resistance in S. typhi is always encoded by plasmids of incompatibility group H (IncH). The complete DNA sequence of the large temperature-sensitive conjugative plasmid R27, the prototype for the IncHI1 family of plasmids, has been compiled and analyzed. This 180 kb plasmid contains 210 open reading frames (ORFs), of which 14 have been previously identified and 56 exhibit similarity to other plasmid and prokaryotic ORFs. A number of insertion elements were found, including the full Tn 10 transposon, which carries tetracycline resistance genes. Two transfer regions, Tra1 and Tra2, are present, which are separated by a minimum of 64 kb. Homologs of the DNA-binding proteins TlpA and H-NS that act as temperature-regulated repressors in other systems have been located in R27. Sequence analysis of transfer and replication regions supports a mosaic-like structure for R27. The genes responsible for conjugation and plasmid maintenance have been identified and mechanisms responsible for thermosensitive transfer are discussed.

Effect of multiple symmetries on the association of R67 DHFR subunits bearing interfacial complementing mutations.

It was shown previously that complementation could be a powerful mean to probe protein-protein interactions in the normally tetrameric R67 DHFR. Indeed, mixing complementing inactive dimeric mutants produced active heterotetramers. This approach turned a homo-oligomer into a hetero-oligomer and thus allowed the use of combinatorial assays, a subtle analysis of the association forces, and a precise determination of the equilibrium dissociation constants (K(D)) by titrimetry. However, for some of the complementing pairs, the experimental data implied multiple equilibria involving heterodimers, although no monomers could be detected. Thus, the reactions involved had to be identified to elaborate a suitable model to determine the K(D) of those pairs correctly. That model suggested that homodimers associated rapidly before the protomers could be redistributed in a multiple equilibrium system. Kinetic data confirmed that view. The association data at equilibrium were analyzed by multiple curve fitting with all plausible combinations of parameters. This gave a confidence interval for K(D) that is safer than the usual 67% or 90% confidence interval. Finally, the K(D) of one specific reaction, the dissociation of a heterotetramer with the relevant symmetry into two homodimers could be determined with the relevant model for each complementing pair, although multiple equilibria were present. These K(D) can thus be used as a set of references data to test and improve theoretical methods such as association free energy calculations.

A spontaneous 99-kb chromosomal deletion results in multi-antibiotic susceptibility and an attenuation of contact haemolysis in Shigella flexneri 2a.

A Tn5-generated mutant (strain S2430) of Shigella flexneri 2a (strain YSH6000) exhibited attenuated virulence and, in addition to the Tn5 insertion in the SalI K fragment of its virulence plasmid, had a 99-kb deletion within its chromosome. Unlike its wild-type parent, strain S2430 was susceptible to ampicillin, streptomycin, tetracycline and chloramphenicol. An independent multi-antibiotic susceptible variant of strain YSH6000 had a similar deletion. Southern blot analysis of pulsed field electrophoresis gels enabled the sizing of this deletion and its mapping to a region of the chromosome on NotI fragment D bounded by the S. flexneri homologues of ompA and pyrC. Hybridisation experiments with a probe specific to the multi-antibiotic resistance region indicated that this large deletion was responsible for antibiotic susceptibility. Both strain S2430 and a derivative of the antibiotic-susceptible variant, with a Tn5 insertion in its SalI K fragment, exhibited an equal reduction in contact haemolysis compared with the Tn5-bearing derivative of strain YSH6000. However, strain S2430 alone clearly displayed delayed plaque forming ability in LLC-MK2 monolayers, suggesting that the two examples of this deletion may not be identical.

Multidrug resistance to antiseptics and disinfectants in coagulase-negative staphylococci.

The occurrence of resistance to antiseptics and disinfectants in clinical isolates of coagulase-negative staphylococci (CNS) was examined. Of 164 clinical strains of CNS isolated in the early 1980s, 65 were resistant to cationic antimicrobial compounds such as cetyltrimethylammonium bromide. Further characterisation of 40 resistant isolates by DNA-DNA hybridisation analysis and phenotypic resistance studies revealed that this resistance was mediated by the multidrug export genes qacA and qacC, characterised previously in Staphylococcus aureus. Of the resistant CNS isolates, 50% contained only qacA, 10% contained only qacC, and the remaining 40% contained both qacA and qacC. Both qacA and qacC genes resided on plasmids in all cases, with qacA located on plasmids of > 10 kb, whereas qacC was located primarily on plasmids of 2-3 kb. Representative qacA and qacC plasmids were characterised by restriction endonuclease mapping, and were found to be similar in some cases, but different in others, to those plasmids on which these genes are found in S. aureus.

Incidence and detection of multi-drug-resistant enterococci in Dublin hospitals.

In February 1994, an outbreak of vancomycin-resistant Enterococcus faecium (VREM) occurred in the oncology unit of a Dublin hospital. Between February and July 1994, VREM was isolated from 18 patients, one staff member and 14 environmental sites within the unit. The isolates also had high-level aminoglycoside and penicillin resistance. Three pulsed-field gel electrophoresis (PFGE) types were identified, two of them from multiple patients and environmental sites. Plasmid typing allowed subdivision of PFGE types. A retrospective study of enterococci isolated from blood cultures between January 1991 and January 1994 showed that, before the outbreak, fewer than 2% of isolates were vancomycin-resistant but that the incidence of high-level gentamicin resistance had increased from 17% to 60% and ampicillin resistance from 22% to 51%. Among clinically significant non-blood-culture enterococci isolated between September and December 1993, fewer than 1% were vancomycin-resistant, 13% were ampicillin-resistant and 44% highly gentamicin-resistant. None produced beta-lactamase. High-content gentamicin disks (120 micrograms) and low-content vancomycin disks (5 micrograms) allowed simple, reliable detection of resistant enterococci. MICs of vancomycin and teicoplanin determined by agar dilution and E-test agreed well, but values tended to be slightly lower by E-test.

A novel plasmid from Staphylococcus epidermidis specifying resistance to kanamycin, neomycin and tetracycline.

The naturally occurring plasmid pSTS7 from Staphylococcus epidermidis mediated resistance to tetracycline via a tetL gene and to kanamycin and neomycin via an aadD gene. Plasmid pSTS7 showed partial restriction map and sequence homology to the previously described tetracycline resistance plasmid pNS1981 from Bacillus subtilis and to the kanamycin/neomycin/bleomycin resistance plasmid pUB110 from S. aureus. Sequence analysis of the regions flanking the two resistance genes in pSTS7 led to the identification of a novel site for interplasmid recombination which could explain the derivation of pSTS7 from the incompatible pNS1981- and pUB110-like parental plasmids under tetracycline-selective pressure.

Antimicrobial resistance and R-factor transfer of salmonellae isolated from chicken carcasses in Greek hospitals.

A total of 62 salmonellae, belonging to six different serotypes, were isolated from 60 out of 87 (69.0%) chicken carcasses delivered to hospitals of Thessaloniki, Greece. Salmonella enteritidis, Salmonella anatum and Salmonella bredeney were the most prevalent serovars. Isolates were examined for antibiotic resistance patterns and R-determinants. Resistance to at least one antibiotic was observed in 36 (58.1%) of them and 18 different resistant profiles were recorded. Nitrofurantoin-resistance was the most common (29.0%), followed by spectinomycin (21.0%), ampicillin (19.4%) and ticarcillin (19.4%). Fourteen (38.9%) of the resistant isolates possessed R-factors and resistance to ampicillin, ticarcillin, trimethoprim and kanamycin was easily self-transferable. However, nitrofurantoin- and spectinomycin-resistance although prevailing, was not found transferable even after mobilization. The high incidence of antibiotic resistant salmonellae among chicken carcasses in our hospital setting suggests the need for public health interventions and possible withdrawal of drug selective pressure.

Frequency of antimicrobial resistance and plasmid profiles of Bacillus cereus strains isolated from milk.

Plasmid profile analysis and susceptibility to eight commonly used antimicrobial agents was done on sixty Bacillus cereus (B. cereus) strains isolated from milk. About seventy two percent (71.7%) of the isolates contained plasmids. The number of plasmids per isolate ranged between 1 and 5, while plasmid sizes ranged between 60 and 0.1 megadaltons (Mda). All isolates were resistant to ampicillin with a relatively high rate of resistance to cotrimoxazole and sulphamethoxazole. The isolates showed a low frequency of resistance to the other antimicrobial agents, with all of them being susceptible to streptomycin. Approximately ninety one (90.7%) percent of the isolates showed multiple resistance. There was no apparent relationship between carriage of plasmids and drug resistance.

Antimicrobial resistance patterns and plasmid survey of Salmonella and Shigella isolated in Ibadan, Nigeria.

Antibiotic resistance patterns among Salmonella and Shigella spp isolated from diarrhoeic human beings were studied. Eleven antibiotic resistance patterns were observed. Plasmid survey of the isolates revealed the presence of multiple plasmids in thirty two of the fifty three strains. More plasmid patterns than antibiotic resistance were observed. Incidence of Salmonellosis and Shigellosis in this environment is caused by genetically unrelated Salmonella and Shigella strains although with identical drug resistance patterns.

Acquired tetracycline resistance genes in nosocomial Salmonella typhimurium infection in a Kenyan hospital.

Tetracyclines have been among the most widely used antibiotics worldwide. Plasmid-mediated tetracycline resistance among hospital strains of bacteria has continued to rise and of major concern has been the transfer of resistance to pathogenic organisms. Bacteraemia due to hospital acquired S. typhimurium has been a major cause of morbidity at Kenyatta National Hospital (KNH), hence the need to study drug susceptibility pattern of this organism. This study also characterized the tetracycline resistance genes using oligonucleotide probes. Ninety seven S. typhimurium strains isolated from patients at KNH were used. Agar dilution method was used to determine minimum inhibitory concentration (MIC). Plasmids were isolated from each strain and the different plasmid profiles were grouped by their molecular weights into 6 patterns. Out of 97, 87 (88%) strains were resistant. MIC ranged from 1 microgram/ml to 128 micrograms/ml. Genes encoding for tetracycline resistance were located on plasmids of molecular weights 65 MDa, 5.2 or both. Plasmid-encoded antimicrobial resistance is likely to spread to other pathogenic organisms, reduce our ability to treat the infection and increase the cost and duration of treatment.

The 120 592 bp IncF plasmid pRSB107 isolated from a sewage-treatment plant encodes nine different antibiotic-resistance determinants, two iron-acquisition systems and other putative virulence-associated functions.

The antibiotic-multiresistance IncF plasmid pRSB107 was isolated by a transformation-based approach from activated-sludge bacteria of a wastewater-treatment plant. It confers resistance to ampicillin, penicillin G, chloramphenicol, erythromycin, kanamycin, neomycin, streptomycin, sulfonamides, tetracycline and trimethoprim and against mercuric ions. Complete sequencing of this plasmid revealed that it is 120 592 bp in size and has a G+C content of 53.1 mol%. The plasmid backbone is composed of three replicons, RepFIA, RepFIB and RepFII, which are almost identical to corresponding regions located on the F-plasmid and on R100. The three replicons encode replication initiation (rep) and replication control, multimer resolution (mrs), post-segregational killing of plasmid-free cells (psk) and active plasmid partitioning (sopABC locus). Part of the F-leading region and remnants of the F-homologous DNA-transfer (tra) module complete the pRSB107 backbone. Plasmid pRSB107 contains a complex, highly mosaic 35 991 bp antibiotic-resistance region consisting of a Tn21- and a Tn10-derivative and a chloramphenicol-resistance module. The Tn21 derivative is composed of a mercury-resistance region (mer), a Tn4352B-like kanamycin/neomycin-resistance transposon, a streptomycin/sulfonamide-resistance module, remnants of the beta-lactam-resistance transposon Tn1, a macrolide-resistance module flanked by copies of IS26 and IS6100, remnants of Tn402 integrating a class 1 integron and the Tn21-specific transposition module. A truncated version of the tetracycline-resistance transposon Tn10 and the chloramphenicol acetyltransferase gene catA complete the pRSB107 resistance region. In addition to antibiotic resistance, pRSB107 encodes the following putative virulence-associated functions: (i) an aerobactin iron-acquisition siderophore system (iuc/iut); (ii) a putative high-affinity Fe(2+) uptake system which was previously identified on a pathogenicity island of Yersinia pestis and in the genome of the phytopathogen Erwinia carotovora subsp. atroseptica SCRI1043; (iii) an sn-glycerol-3-phosphate transport system (ugp); and (iv) the virulence-associated genes vagCD having a possible function in stable plasmid inheritance. All the accessory modules are framed by insertion sequences, indicating that pRSB107 was gradually assembled by integration of different horizontally acquired DNA segments via transposition or homologous recombination.