Brief report: diagnosis of Whipple's disease by molecular analysis of peripheral blood.
Explore the source record for details and available documents.
Adult↗
Biomedical subjects
G L Archer
Publications and source records attributed to G L Archer.
Origin and evolution of DNA associated with resistance to methicillin in staphylococci.
The gene mediating resistance to methicillin in staphylococci (mecA) and its flanking sequences (mec DNA) make up a chromosomal DNA locus that is unique to methicillin-resistant bacteria; no equivalent locus exists in methicillin-susceptible cells. The origin of mec DNA is not known, but evidence supports horizontal transfer of mec DNA between different staphylococcal species and of the mecA gene between different Gram-positive genera.
Chromosome Mapping↗
Coagulase-negative staphylococci: pathogens associated with medical progress.
Coagulase-negative staphylococcal bacteremia and infections of prosthetic medical devices have become major clinical problems. Efforts to differentiate contaminating from infecting isolates consume the time of microbiology laboratory personnel; decisions over when and with what to institute therapy for multiresistant isolates consume the energy of clinicians; and the need to institute expensive parenteral antimicrobial therapy consumes the hospital pharmacy budget. It is clear that the increased incidence of coagulase-negative staphylococcal infections is the result of medical progress and is due to the use of invasive and indwelling medical devices. Multiresistant organisms have evolved that will survive in the presence of antimicrobial agents designed to eradicate more traditional pathogens. They have an ecological niche on human skin from which they are difficult to eradicate, and they have adapted themselves to survive on inert devices designed to persist indefinitely in the human body. Since it is likely that the use of prosthetic medical devices will continue to increase, we need to device innovative strategies for the diagnosis, treatment, and prevention of infections of these indwelling foreign bodies. Studies that will address these issues should be a major goal of future research on hospital-acquired infections.
Bacteremia↗
Antimicrobial susceptibility of coagulase-negative staphylococci.
Explore the source record for details and available documents.
Anti-Bacterial Agents↗
Dissemination among staphylococci of DNA sequences associated with methicillin resistance.
DNA probes consisting of pUC19 containing cloned Staphylococcus aureus chromosomal fragments were constructed from two methicillin-resistant S. aureus strains with different DNA sequences 5' to mecA, the gene that mediates methicillin resistance. The probe from one strain, BMS1, contained a portion of the regulatory sequences (the terminal 641 bp of mecR1 and all of mecI) associated with the induction and repression of mecA transcription (pGO195). The second probe, from strain COL (pGO198), contained DNA not found in strain BMS1. This DNA was within the sequences added at the site of a mecR1 deletion. Genomic digests of 14 S. aureus isolates recovered between 1961 and 1969 all hybridized with pGO198. In contrast, 78% (36 of 46) of the S. aureus organisms isolated since 1988 hybridized with pGO195 but not with pGO198; the remainder hybridized with pGO198. No S. aureus isolates hybridized with both probes. Staphylococcus epidermidis digests hybridized with pGO198 (46%), pGO195 (14%), or both probes (35%); all 20 Staphylococcus haemolyticus isolates hybridized with pGO198. The restriction fragment length polymorphism patterns of all pGO198-hybridizing regions in S. aureus were identical to those in strain COL. In addition, the mecR1 deletion junction nucleotide sequences of eight S. aureus and six S. epidermidis isolates were identical. However, 21 of 23 S. epidermidis and all 20 S. haemolyticus isolates had from 5 to more than 20 additional chromosomal bands that hybridized with pGO198; none of 21 S. aureus isolates had additional hybridizing bands. These data suggest that the additional DNA responsible for the mecR1 deletion was part of a repetitive, and possibly mobile, element resident in coagulase-negative staphylococci but not in S. aureus. These data also support a hypothesis that the deletion event occurred in a coagulase-negative staphylococcus with subsequent acquisition of the interrupted sequences by S. aureus.
Base Sequence↗
Transcriptional regulation by TrsN of conjugative transfer genes on staphylococcal plasmid pGO1.
The major conjugative transfer gene cluster of staphylococcal plasmid pGO1 (trs) consists of 13 open reading frames (trsA to trsM) transcribed from one DNA strand and a single 189-bp open reading frame (trsN) within the first 348 bp of trs that is transcribed divergently. Promoter regions for trsN and trsA partially overlap. TrsN, a 7,181-Da protein, was purified as a fusion to glutathione S-transferase and found to have DNA-binding activity. Increasing concentrations of the fusion protein progressively retarded the gel migration of PCR-generated DNA fragments containing predicted promoters 5' to trsL, trsA, and trsN. The target sequences contained areas of identity, including regions of dyad symmetry, that were protected in DNase I footprinting studies. The binding of TrsN to its trsL target was required for this target DNA to be stably introduced into Staphylococcus aureus on a high-copy-number vector. Provision of excess TrsN from this high-copy-number vector in S. aureus decreased beta-galactosidase activity from a trsL-lacZ transcriptional fusion and decreased pGO1 conjugation frequency. Conversely, both transcription and conjugation increased in the presence of excess trsL target. We propose that TrsN negatively regulates the transcription of genes essential for conjugative transfer by binding to regions 5' to their translational start sites.
Amino Acid Sequence↗
DNA sequence and units of transcription of the conjugative transfer gene complex (trs) of Staphylococcus aureus plasmid pGO1.
The conjugative transfer genes of 52-kb staphylococcal R plasmid pGO1 were localized to a single BglII restriction fragment and cloned in Escherichia coli. Sequence analysis of the 13,612-base transfer region, designated trs, identified 14 intact open reading frames (ORFs), 13 of which were transcribed in the same direction. Each ORF identified was preceded by a typical staphylococcal ribosomal binding sequence, and 10 of the 14 proteins predicted to be encoded by these ORFs were seen when an E. coli in vitro transcription-translation system was used. Functional transcription units were identified in a Staphylococcus aureus host by complementation of Tn917 inserts that abolished transfer and by Northern (RNA) blot analysis of pGO1 mRNA transcripts. These studies identified three complementation groups (trsA through trsC, trsD through trsK, and trsL-trsM) and four mRNA transcripts (trsA through trsC [1.8 kb], trsA-trsB [1.3 kb], trsL-trsM [1.5 kb], and trsN [400 bases]). No definite mRNA transcript was seen for the largest complementation group, trsD through trsK (10 kb). Comparison of predicted trs-encoded amino acid sequences to those in the data base showed 20% identity of trsK to three related genes necessary for conjugative transfer of plasmids in gram-negative species and 32% identity of trsC to a gene required for conjugative mobilization of plasmid pC221 from staphylococci.
Amino Acid Sequence↗
Mobilization of recombinant plasmids from Staphylococcus aureus into coagulase negative Staphylococcus species.
pC221, a small nonconjugative staphylococcal plasmid, can be mobilized between staphylococci by pG01, a larger conjugative plasmid. pC221 carries the two transacting genes, mobA and mobB, which are needed for its mobilization. The products of these genes create a site-specific single-stranded nick (mobA) and then facilitate DNA transfer (mobB). Several useful Escherichia coli-staphylococcal shuttle plasmids containing the cloned single-stranded nick site were created and successfully mobilized into Staphylococcus aureus and two coagulase-negative staphylococci, S. epidermidis and S. saprophyticus, by providing mob genes (pC221) and conjugative transfer genes (pG01) in trans in the donor. These vectors may offer a genetic system for the introduction of recombinant plasmids into coagulase negative staphylococci.
Bacterial Proteins↗
Hemagglutination and adherence to plastic by Staphylococcus epidermidis.
Staphylococcus epidermidis is an important nosocomial pathogen responsible for intravenous catheter-related bacteremia and infections of other prosthetic medical devices. We found that the ability of S. epidermidis to hemagglutinate erythrocytes correlated with the adherence of bacteria to plastic and to intravenous catheters. S. epidermidis isolates responsible for prosthetic-valve endocarditis (n = 61) and isolates from intravenous catheters (n = 59) were significantly more likely to cause hemagglutination than isolates from the skin of preoperative cardiac surgery patients (n = 19) (P = 0.027). S. epidermidis isolates (n = 23) recovered from the skin of patients 7 to 10 days after cardiac surgery were significantly more likely to exhibit hemagglutination than the preoperative isolates (P = 0.015). By a quantitative adherence assay, we also observed that the hemagglutination titer and number of species of erythrocytes agglutinated correlated directly with adherence to polystyrene (P less than 0.001). In addition, hemagglutinating isolates were significantly more likely to be recovered in high number from intravenous catheters when semiquantitative catheter culture techniques were used (P less than 0.001). We speculate that hemagglutinin(s) either plays a direct role in adherence to polymers and thus prosthetic-device infection or serves as an easily demonstrable marker for adherence-prone isolates.
Animals↗
Colonization of the female genital tract with Staphylococcus saprophyticus.
The prevalence of colonization by Staphylococcus saprophyticus of the urogenital tracts of 276 women from an outpatient gynecology practice was determined by using selective and enrichment culture techniques. Nineteen subjects (6.9%) were found to be colonized by S. saprophyticus. The rectum was the most frequent site of colonization and was responsible for 40% of the isolates; this was followed in decreasing order by the urethra, urine, and cervix. Women colonized by S. saprophyticus were more likely to have experienced a urinary tract infection in the previous 12 months (P = 0.058; odds ratio, 2.844; 95% confidence interval, 1.054 to 7.671). Patients colonized by S. saprophyticus tended to have had their menstrual periods more recently (P = 0.066), experienced sexual intercourse more recently (P = 0.168), and had a recent or concurrent diagnosis of vaginal candidiasis (P = 0.111; odds ratio, 2.393; 95% confidence interval, 0.877 to 6.528). A seasonal variation in colonization was observed, with colonization most likely occurring during the summer and fall. Follow-up for an average of 6.75 months failed to document any colonized woman progressing to symptomatic urinary tract infection. In addition, 21 women colonized by non-S. saprophyticus, novobiocin-resistant, coagulase-negative staphylococci were identified and characterized.
Adult↗
New mechanisms of bacterial resistance to antimicrobial agents.
Explore the source record for details and available documents.
Anti-Bacterial Agents↗
Conjugative transfer genes in staphylococcal isolates from the United States.
Staphylococcus aureus and coagulase-negative staphylococcal isolates from various geographic areas in the United States were examined by using a conjugative transfer gene DNA probe in dot-blot hybridization assays. Of 175 S. aureus isolates, 47 (27%) hybridized with the probe, while 24 of 208 (11.5%) coagulase-negative staphylococci hybridized. However, among methicillin-resistant S. aureus 52% (45 of 89) were probe positive while only 2% (2 of 86) of methicillin-susceptible S. aureus were probe positive. In contrast, 12.5% (22 of 176) of methicillin-resistant and 6% (2 of 32) of methicillin-susceptible coagulase-negative staphylococci contained transfer genes. All but one of the staphylococci containing transfer genes were resistant to gentamicin; 91.5% of S. aureus and 65% of coagulase-negative staphylococci containing transfer genes transferred gentamicin resistance to a S. aureus recipient. Of the 12 isolates that hybridized with the probe but did not transfer resistance, 10 (6 coagulase-negative staphylococci and 4 S. aureus) carried both gentamicin resistance and conjugative transfer genes on the same plasmid. Of these 10, 6 contained plasmid target fragments of sizes different from that of the probe, suggesting additions or deletions of DNA essential for transfer, while in 4 no such alterations could be detected. In two coagulase-negative staphylococci the entire transfer region was apparently integrated into the chromosome. Thus, staphylococci carrying conjugative transfer genes are widely disseminated in the United States and are usually found in multiresistant isolates on plasmids that also encode gentamicin resistance.
Anti-Bacterial Agents↗
Detection of methicillin resistance in staphylococci by using a DNA probe.
A DNA probe derived from the PBP 2a gene of the methicillin-resistant Staphylococcus aureus COL was compared with phenotypic microbiologic tests for its ability to identify methicillin-resistant and -susceptible staphylococci. Lysates were applied to nitrocellulose with a dot blot apparatus. Isolates tested were both S. aureus and coagulase-negative staphylococci that had been recovered from a variety of geographic and clinical sources. When compared with a spread plate phenotypic test, the DNA probe gave sensitivity, specificity, and predictive values for both positive and negative tests of 100% for 204 S. aureus isolates (103 positive, 101 negative) and 99, 95, 99, and 95%, respectively, for 249 coagulase-negative staphylococci (210 positive, 39 negative). The probe was more sensitive than broth microdilution and more specific than agar dilution in identifying methicillin-resistant and -susceptible coagulase-negative staphylococci; all tests were equally accurate in identifying the methicillin susceptibility of S. aureus. DNA probe analysis for determining the methicillin susceptibility of staphylococci was rapid, easily interpretable, and equally accurate with radioactive and nonradioactive probes, and it gave results equivalent to the most sensitive microbiologic test for all staphylococcus species studied.
Bacterial Proteins↗
Antimicrobial resistance in nosocomial isolates of Staphylococcus haemolyticus.
Staphylococcus haemolyticus is frequently cultured from hospitalized patients and is characterized by resistance to multiple antimicrobial agents. We found that S. haemolyticus represented 70 of 524 (13%) coagulase-negative staphylococcal isolates identified by the clinical microbiology laboratories of two hospitals over 2 months. S. haemolyticus isolates were recovered from wounds (44%), urine (26%), blood (10%), and other sources (20%). All S. haemolyticus isolates were tested for susceptibility to six antimicrobial agents; 77% were resistant to three or more agents, and 41% were resistant to five or six agents. In addition, among 47 multiply resistant isolates, high MICs (greater than or equal to 6.25 micrograms/ml) of vancomycin (62% of isolates) and teicoplanin (91% of isolates) were found. DNA probes which were derived from S. epidermidis or S. aureus and which contained sequences associated with resistance to antimicrobial agents were used to detect specific genes in the total cellular and plasmid DNAs of 10 resistant S. haemolyticus isolates. Resistance gene probes and the numbers of resistant isolates hybridizing were as follows: methicillin, 10 of 10; gentamicin, 9 of 10; erythromycin, 7 of 10; and trimethoprim, 0 of 10. Genes for resistance to methicillin were found only in chromosomal locations, genes for resistance to gentamicin were found in both chromosomal and plasmid locations, and genes for resistance to erythromycin were found in plasmid locations only. With the exception of trimethoprim resistance determinants, similar genes were found among concurrently isolated multiply resistant S. epidermidis isolates from our hospitals. S. haemolyticus is a potentially important nosocomial species which readily acquires antimicrobial resistance genes and which shares, to some extent, in a common gene pool with S. epidermidis.
Anti-Bacterial Agents↗
Mobility of gentamicin resistance genes from staphylococci isolated in the United States: identification of Tn4031, a gentamicin resistance transposon from Staphylococcus epidermidis.
Homologous genes encoding resistance to gentamicin, tobramycin, and kanamycin through the bifunctional acetylating [AAC(6')] and phosphorylating [APH(2")] aminoglycoside-modifying enzyme were identified in staphylococci isolated from patients in the United States. The mobility of gentamicin resistance (Gmr) genes found on a prototype conjugative plasmid (pGO1) was compared with that of genes cloned from chromosomal sites. Plasmid-encoded Gmr genes and flanking sequences were introduced onto a temperature-sensitive plasmid (pRN3208) from pGO1 by homologous recombination between insertion sequence-like elements present on both replicons. Growth of Staphylococcus aureus strains containing the temperature-sensitive recombinant (pGO161) at the nonpermissive temperature for plasmid replication (42 degrees C) revealed no translocation of Gmr from its plasmid location. A transposon (Tn551) resident on the same replicon did translocate. Chromosomal Gmr determinants were cloned, together with the gene for trimethoprim resistance (dfrA), from three geographically distinct S. epidermidis isolates; two were subcloned onto temperature-sensitive Escherichia coli-S. aureus shuttle plasmids as 7.2-kilobase BglII fragments. Growth of both recombination-deficient and-proficient S. aureus strains containing the cloned genes at 42 degrees C allowed detection of transposition of Gmr sequences and identification of insertion into random chromosomal sites. We have designated this 5-kilobase transposon from S. epidermidis as Tn4031.
Cloning, Molecular↗
Identification and cloning of the conjugative transfer region of Staphylococcus aureus plasmid pGO1.
The conjugative transfer (tra) genes of a 52-kilobase (kb) staphylococcal plasmid, pGO1, were localized by deletion analysis and transposon insertional inactivation. All transfer-defective (Tra-) deletions and Tn551 or Tn917 transposon insertions occurred within a 14.5-kb BglII fragment. Deletions and insertions outside this fragment all left the plasmid transfer proficient (Tra+). The tra region was found to be flanked by directly repeated DNA sequences, approximately 900 base pairs in length, at either end. Clones containing the 14.5-kb BglII fragment (pGO200) and subclones from this fragment were constructed in Escherichia coli on shuttle plasmids and introduced into Staphylococcus aureus protoplasts. Protoplasts could not be transformed with pGO200E (pGO200 on the staphylococcal replicon, pE194) or subclones containing DNA at one end of the tra fragment unless pGO1 or specific cloned tra DNA fragments were present in the recipient cell. However, once stabilized by sequences present on a second replicon, each tra fragment could be successfully introduced alone into other plasmid-free S. aureus recipients by conjugative mobilization or transduction. In this manner, two clones containing overlapping fragments comprising the entire 14.5-kb BglII fragment were shown to complement each other. The low-frequency transfer resulted in transconjugants containing one clone intact, deletions of that clone, and recombinants of the two clones. The resulting recombinant plasmid (pGO220), which regenerated the tra region intact on a single replicon, transferred at frequencies comparable to those of pGO1. Thus, all the genes necessary and sufficient for conjugative transfer of pGO1 are contained within a 14.5-kb region of DNA.
Cloning, Molecular↗
Mobilization of the relaxable Staphylococcus aureus plasmid pC221 by the conjugative plasmid pGO1 involves three pC221 loci.
The Staphylococcus aureus plasmid pC221, a 4.6-kilobase multicopy chloramphenicol resistance plasmid that forms plasmid-protein relaxation complexes, was mobilized for transfer by the conjugative plasmid pGO1. Two open reading frames on the pC221 genome, now designated mobA and mobB, as well as a cis-acting locus, the putative oriT, were shown to be in involved in pC221 mobilization. The mobA (but not mobB) and oriT loci were required for pC221 relaxation, and relaxation was necessary but not sufficient for pC221 mobilization by pGO1. oriT was cloned onto a pE194 derivative and complemented in trans for both relaxation and mobilization. Mobilization of relaxable plasmids in S. aureus appears to be analogous to mobilization by donation observed in gram-negative bacteria.
Conjugation, Genetic↗
Restriction enzyme analysis of plasmid DNA and bacteriophage typing of paired Staphylococcus aureus blood culture isolates.
We compared restriction enzyme analysis of plasmid (REAP) DNA profiling with bacteriophage typing for determination of similarities and differences among 50 pairs of Staphylococcus aureus blood isolates from patients with multiple positive blood cultures. Isolates from 17 pairs did not have detectable plasmids. Isolates from 33 pairs had plasmids classified into 17 distinct REAP DNA profiles. Paired isolates from 31 of these episodes were identical to one another. By phage typing, 35 pairs had strong lytic reactions to a phage(s), 9 pairs lacked strong reactions, and 6 pairs consisted of a strongly reactive isolate and an isolate with no strong reaction to a phage. When consolidated into 11 general phage groups, pairs from 44 of the 50 episodes were in the same general group. REAP DNA profiles were highly reproducible (99%), whereas phage typing was not. REAP DNA profiling is superior to phage typing as a technique for determining similarities and differences among S. aureus blood isolates.
Bacteriophage Typing↗