Examination of osteoblast-orthopaedic biomaterial interactions using molecular techniques.

Molecular techniques can be used to elucidate the effects of extended periods of cell-biomaterial interactions on the time-course and level of expression of particular genes which determine cellular phenotype. We used the polymerase chain reaction to demonstrate the expression of genes for the bone-related proteins osteocalcin, osteonectin and osteopontin by neonatal rat calvarial osteoblasts. In addition, Northern blotting was subsequently used to show that messenger RNAs encoding osteonectin and osteopontin were consistently expressed during a 5 wk period of interaction of osteoblasts with Ti-6Al-4V, a commercial brand of hydroxyapatite, and tissue culture polystyrene.

Relatedness of coagulase-negative staphylococci causing bacteremia in low-birthweight infants.

OBJECTIVE: To investigate coagulase-negative staphylococcus (CONS) causing bacteremia in a neonatal intensive care unit (NICU). DESIGN: A 14-month retrospective review of 47 infants in the NICU with CONS bacteremia was undertaken to determine CONS glycocalyx production, plasmid pattern, total DNA restriction fragment polymorphism, and clinical risk factors. RESULTS: The isolates included 32 Staphylococcus epidermidis, six Staphylococcus haemolyticus, four Staphylococcus warneri, four Staphylococcus saprophyticus, and one Staphylococcus hominis. Sixty-five percent of S epidermidis produced glycocalyx; other species did not. Oxacillin resistance (52%) and the antibiograms of the CONS were consistent with other units in the hospital. Five similar CONS plasmid patterns were found among 16 isolates; 31 isolates had unique patterns. Extractions of total DNA from these isolates were digested using HindIII, HaeIII, and BstEII. Those with similar restriction fragment length patterns could not linked as nosocomially transmitted among infants with bacteremia. CONCLUSION: Our observations suggest that multiple strains of CONS infect infants in the NICU who have similar risk factors. Although current infection control practices limit transmission of a pathogen, they do not prevent CONS bacteremias.

Recurrent bacterial peritonitis caused by Neisseria cinerea in a chronic ambulatory peritoneal dialysis (CAPD) patient.

We present an unusual case of recurrent (chronic ambulatory peritoneal dialysis) CAPD-associated peritonitis caused by Neisseria cinerea. Using DNA restriction fragment length polymorphism (RFLP) analysis, we determined that the recurrent infection was caused by reinfection with a different N. cinerea strain rather than relapse with the index strain and that the probable origin of the reinfecting organism was the patient's upper respiratory tract.

Molecular analysis of an acatalasemic mouse mutant.

The Csb acatalasemia mouse mutant differentially expresses reduced levels of catalase activity in a tissue specific manner. In order to pinpoint the molecular lesion that imparts the acatalasemia phenotype in Csb mice we have utilized the polymerase chain reaction technique to isolate catalase cDNA clones from control and Csb mouse strains. Sequence analyses of these cDNA clones have revealed a single nucleotide difference within the coding region of catalase between control and Csb mice. This nucleotide transversion (G----T) is located in the third position of amino acid 11 in the catalase monomer. In control mouse strains glutamine (CAG) is encoded at amino acid 11, while in Csb mice this codon (CAT) encodes histidine. This amino acid is located within a region that forms the first major alpha-helix in the amino-terminal arm of the catalase subunit and, as such, may render the catalase molecule unstable under certain physiological conditions.

Restriction map and properties of Klebsiella oxytoca plasmid pACM1.

pACM1 is a conjugative multiresistance (putative IncM) plasmid from Klebsiella oxytoca. In order to make a structural and functional map, cloned fragments of pACM1 were systematically isolated from pUC19 libraries using DNA probes from previously cloned fragments. All but approximately 3.6 kb of the plasmid were cloned and a consensus map is presented. Certain pACM1 fragments were "unclonable" (i.e., could not be detected among transformants) unless a 7-kb KpnI fragment was also present in the recombinant construct. Restriction sites found in a portion of the 7-kb KpnI fragment resemble those of the iml determinant region of IncM plasmid R446; therefore, the 7-kb fragment is probably within or includes part of the IncM tra (conjugation) operon. It is probable that pACM1 has loci functionally similar to the kil (lethal) and kor (kill override) loci in the tra operons of IncN or IncP plasmids. pACM1 can be a valuable model for the study of IncM plasmids.

Nucleotide sequence of the chromosomal ampC gene of Enterobacter aerogenes.

The AmpC beta-lactamase gene and a small portion of the regulatory ampR sequence of Enterobacter aerogenes 97B were cloned and sequenced. The beta-lactamase had an isoelectric point of 8 and conferred cephalosporin and cephamycin resistance on the host. The sequence of the cloned gene is most closely related to those of the ampC genes of E. cloacae and C. freundii.

Nucleotide sequence of a 7-kb fragment of pACM1 encoding an IncM DNA primase and other putative proteins associated with conjugation.

A 7-kb fragment of pACM1 (fragment 90¿91) containing one or more kor (kill-override) loci was sequenced, and 28 open reading frames (ORFs; >/=50 codons) were identified. The nucleotide sequence has no significant homologs in the GenBank database except for a 1.3-kb region 98.6% identical to the iml (insensitivity to phage PhiM-mediated lysis) determinant fragment of IncM plasmid R446. Deduced amino acid sequences for several ORFs are homologous to those of known proteins, including the Sog DNA primases of IncI1 plasmids R64 and ColIb-P9 and the TraL, TraM, and TraN products of ColIb-P9. Two protein products of the putative primase ORF (ORF 1, 1100 amino acids) were detected by SDS-PAGE. The 158- and 107-kDa proteins were designated PriL and PriS, respectively. PriS is apparently produced by an in-frame reinitiation of the ORF 1 transcript at a second start codon located between a Sau96I site and a PstI site. The motif EGYATA, conserved among primases and associated with primase function, occurs in the first one-third of the deduced amino acid sequence of PriL and is not included in PriS. Partial suppression of the temperature-sensitive dnaG3 mutation in BW86 was demonstrated by recombinants that overexpressed both PriL and PriS, but not by constructs overexpressing only PriS. Therefore, primase function can be assigned to PriL. Fragment 90/91 represents a portion of the IncM tra region, which has not previously been examined in detail.

The cassettes and 3' conserved segment of an integron from Klebsiella oxytoca plasmid pACM1.

pACM1 is a conjugative multiresistance plasmid from Klebsiella oxytoca that encodes SHV-5 extended-spectrum beta-lactamase (ESBL) and has two integrons. The first is a type I (sul type); the second, detected by hybridization with an intI gene probe, has been putatively identified as a defective type I integron. The cassette region of the first integron has now been fully sequenced and contains three aminoglycoside resistance determinants (aac(6')-Ib, aac(3)-Ia, and ant(3")-Ia) and two open reading frames of unknown function. In addition, sequencing of a region downstream from the qacEDelta1-sulI-ORF 5 gene cluster of the first integron revealed a copy of insertion sequence IS6100 flanked by inverted copies of sequence from the 11.2-kb insert (In2) of Tn21. This arrangement is similar to that found in In4 of Tn1696. The coincidence of an ESBL gene and mobile elements on a conjugative plasmid has potential implications for the spread of ESBL-mediated drug resistance, though evidence of bla((SHV-5)) movement mediated by these elements has not been found.

Increased oxacillin activity associated with glycopeptides in coagulase-negative staphylococci.

Vancomycin resistance in methicillin-resistant staphylococci presents a potential therapeutic problem. In order to understand the impact of low-level vancomycin resistance in coagulase-negative staphylococci, stepwise selection of vancomycin resistance was accomplished by growing Staphylococcus haemolyticus in culture media with increasing concentrations of vancomycin. A >40-fold increase in susceptibility to beta-lactam antibiotics was observed. No obvious alterations in the growth curve, the presence of the mecA gene, total DNA restriction fragment length polymorphism (RFLP), beta-lactamase production, or the crude protein fraction were detected in the Staphylococcus haemolyticus-derived clones when compared to the original isolate. The proportion of the oxacillin-heteroresistant population also remained similar. A comparable phenomenon occurred with the selection of Staphylococcus epidermidis exhibiting low-level resistance to vancomycin. Additionally, it was observed that clinical isolates of coagulase-negative staphylococci grown in the presence of sub-minimum inhibitory concentrations of either vancomycin or teicoplanin lost their high-level resistance to oxacillin. Checkerboard tests showed that the combination of vancomycin and oxacillin was synergistic for two isolates of Staphylococcus haemolyticus, two of four isolates of Staphylococcus epidermidis, and one isolate of Staphylococcus hominis.

Selection of high-level oxacillin resistance in heteroresistant Staphylococcus aureus by fluoroquinolone exposure.

To study the effect of fluoroquinolone exposure on the expression of mec(A)-encoded oxacillin resistance, population analysis profiling was performed on four strains of fluoroquinolone-susceptible, mec(A)-positive, heteroresistant Staphylococcus aureus. Growth in the presence of 0.5 x MIC of a fluoroquinolone resulted in >10-fold increase in the proportion of the population that grew on agar containing oxacillin 128 mg/L. Ciprofloxacin exhibited a greater effect than moxifloxacin, levofloxacin and gatifloxacin (average 3400-, 220-, 170- and 49-fold increase in oxacillin-resistant colonies versus the control, respectively). The increase was directly proportional to the fluoroquinolone concentration and could be detected as early as 8 h after exposure to the fluoroquinolone. At 8 h, the absolute number of colonies that grew on oxacillin 128 mg/L was similar whether or not the isolate was exposed to the fluoroquinolone, but the total cfu on non-selective media decreased. The resultant oxacillin-resistant colonies also showed a 1.5- to 3-fold increase in fluoroquinolone MIC. No oxacillin resistance was observed on two similarly treated fluoroquinolone-susceptible, mec(A)-negative strains. It appears that fluoroquinolones influence oxacillin resistance by selective inhibition or killing of the more susceptible subpopulations in heteroresistant S. aureus. The surviving populations are more resistant to both oxacillin and fluoroquinolone. The mechanisms of resistance to the two agents may be unrelated but tend to be associated. This could explain in part the observed increases in fluoroquinolone-resistant MRSA.

The resistance and integrase genes of pACM1, a conjugative multiple-resistance plasmid, from Klebsiella oxytoca.

pACM1 is an 85-kb conjugative plasmid from a clinical isolate of Klebsiella oxytoca that encodes resistance to beta-lactams (mediated by SHV-5 extended spectrum beta-lactamase), trimethoprim, sulfonamides, tetracycline, aminoglycosides, and mercuric chloride. The expression of the aminoglycoside resistance is difficult to detect, which could have clinical implications. A region of pACM1 containing five resistance genes and two putative integrons was characterized by restriction mapping and partial DNA sequencing. One integron appears to be class I (sull type); the second lacks a recognizable 3' conserved segment. Neither integron has the BamHI site predicted for the 5' conserved segment. Plasmids encoding SHV-5 from other bacterial strains appear to be closely related to pACM1 by restriction enzyme analysis, but have resistance/ integron regions that vary in size and content from that of pACM1. Integrase-mediated recombination might be responsible for genetic divergence in a widely distributed family of pACM1-like plasmids.

Molecular epidemiology of an SHV-5 extended-spectrum beta-lactamase in enterobacteriaceae isolated from infants in a neonatal intensive care unit.

Klebsiella oxytoca that produced extended-spectrum beta-lactamase (ESBL) and were resistant to ceftazidime were isolated from infants in a neonatal intensive care unit (NICU). During a 30-week period, 3 infants developed infections and an additional 60 infants were colonized with these bacteria. The molecular typing data suggested transmission of a single strain of ceftazidime-resistant K. oxytoca among 48 of the 63 infants. The ESBL of 46 of the 48 similar isolates, 14 of the remaining 15 isolates, and 6 other Enterobacteriaceae appeared to be associated with a conjugative plasmid of approximately 85 kb. The ESBL gene was cloned, and DNA sequencing confirmed that the ESBL was an SHV-5. Hybridization data suggested that the SHV-5 gene was transmitted to other Enterobacteriaceae in vivo. The spread of the ESBL was reduced through adherence to infection control practices.