The isolation of adult rat liver parenchymal cells

Summary This report describes procedures for the isolation and maintenance of monolayer culture of adult rat liver hepatic parenchymal cells. Isolation of the cells is accomplished using perfusion in situ with a calcium-free buffer followed by buffered collagenase. Gravity sedimentation and selective media are used to limit the contribution of nonparenchymal cells in the cultures.     

Lesions of the MPO or AV3V: influences on fluid intake

Electrolytic lesions in the MPO of rats had no significant effects on ad lib food and water intake, but impaired the drinking response to 1 M NaCl. Large MPO lesions also produced a persistent increase in plasma osmolality. In Experiment 2, we depleted neurons from the MPO of rats by iontophoretic application of the neurotoxin kainic acid (KA) which destroys nerve cell bodies without damage to fibers of passage. KA-induced neuron depletion in the MPO of rats significantly reduced the drinking response to 1.0 M saline, to 30% PG, and to 30 micrograms/kg isoproterenol. Ad lib water intake and drinking responses to food or water deprivation, to low concentrations (0.5 M) of hypertonic saline, to low concentrations (10% or 20%) of PG, and to systemic administration of 1.5 mg/kg angiotensin II were within the normal range. In Experiment 3, rats with electrolytic lesions that were strictly confined to the tissue immediately surrounding the wall of the anteroventral portion of the third ventricle (AV3V), without invading the MPO displayed normal ad lib food and water intake and plasma osmolality as well as drinking responses to water deprivation, hypertonic saline (0.5 or 1.0 M), angiotensin II (1.5 mg/kg) and isoproterenol (30 micrograms/kg).     

Characterization of clinical isolates of Klebsiella pneumoniae from 19 laboratories using the National Committee for Clinical Laboratory Standards extended-spectrum beta-lactamase detection methods

Extended-spectrum beta-lactamases (ESBLs) are enzymes found in gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam. In 1999, the National Committee for Clinical Laboratory Standards (NCCLS) published methods for screening and confirming the presence of ESBLs in Klebsiella pneumoniae, Klebsiella oxytoca, and Escherichia coli. To evaluate the confirmation protocol, we tested 139 isolates of K. pneumoniae that were sent to Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) from 19 hospitals in 11 U.S. states. Each isolate met the NCCLS screening criteria for potential ESBL producers (ceftazidime [CAZ] or cefotaxime [CTX] MICs were > or =2 microg/ml for all isolates). Initially, 117 (84%) isolates demonstrated a clavulanic acid (CA) effect by disk diffusion (i.e., an increase in CAZ or CTX zone diameters of > or =5 mm in the presence of CA), and 114 (82%) demonstrated a CA effect by broth microdilution (reduction of CAZ or CTX MICs by > or =3 dilutions). For five isolates, a CA effect could not be determined initially by broth microdilution because of off-scale CAZ results. However, a CA effect was observed in two of these isolates by testing cefepime and cefepime plus CA. The cefoxitin MICs for 23 isolates that failed to show a CA effect by broth microdilution were > or =32 microg/ml, suggesting either the presence of an AmpC-type beta-lactamase or porin changes that could mask a CA effect. By isoelectric focusing (IEF), 7 of the 23 isolates contained a beta-lactamase with a pI of > or =8.3 suggestive of an AmpC-type beta-lactamase; 6 of the 7 isolates were shown by PCR to contain both ampC-type and bla(OXA) genes. The IEF profiles of the remaining 16 isolates showed a variety of beta-lactamase bands, all of which had pIs of < or =7.5. All 16 isolates were negative by PCR with multiple primer sets for ampC-type, bla(OXA), and bla(CTX-M) genes. In summary, 83.5% of the K. pneumoniae isolates that were identified initially as presumptive ESBL producers were positive for a CA effect, while 5.0% contained beta-lactamases that likely masked the CA effect. The remaining 11.5% of the isolates studied contained beta-lactamases that did not demonstrate a CA effect. An algorithm based on phenotypic analyses is suggested for evaluation of such isolates.     

Evaluation of the Vitek GPS-TA card for laboratory detection of high-level gentamicin and streptomycin resistance in enterococci.

The Vitek GPS-TA card (Vitek Systems, Hazelwood, Mo.) was compared with single-concentration broth microdilution and disk diffusion methods using high-content disks for the detection of high-level resistance to gentamicin and streptomycin in 99 isolates of enterococci (81 Enterococcus faecalis isolates and 18 Enterococcus faecium isolates). The GPS-TA card accurately detected high-level resistance to gentamicin, but not streptomycin, in E. faecalis. When streptomycin is being considered for therapy, either disk diffusion or time-kill studies should be used to confirm susceptible results obtained by Vitek testing. Additional studies are needed to determine the best method for testing E. faecium isolates.     

Reproducibility of interpretation of the test for antibody-coated bacteria in urinary sediment.

The direct immunofluorescence technique for detecting antibody-coated bacteria in urinary sediment is felt to be useful in distinguishing infection of the kidney from infection of the bladder. An independent, blind multiple-reading system was used to measure interobserver variability in the evaluation of slides of urinary sediments for antibody-coated bacteria. Three independent observers agreed unanimously on first reading in 88% of 253 specimens. When compared with the majority opinion, the sensitivity and specificity of an individual reading were 91 and 95%, respectively.     

Microbicides 2006 conference

Current HIV/AIDS statistics show that women account for almost 60% of HIV infections in Sub-Saharan Africa. HIV prevention tools such as male and female condoms, abstinence and monogamy are not always feasible options for women due to various socio-economic and cultural factors. Microbicides are products designed to be inserted in the vagina or rectum prior to sex to prevent HIV acquisition.     

Optimization of computer software settings improves accuracy of pulsed-field gel electrophoresis macrorestriction fragment pattern analysis

Computer-assisted analysis of pulsed-field gel electrophoresis (PFGE) libraries can facilitate comparisons of fragment patterns present on multiple gels. We evaluated the ability of the Advanced Analysis (version 4.01) and Database (version 1.12) modules of the Phoretix gel analysis software package (Nonlinear USA, Inc., Durham, N.C.) to accurately match DNA fragment patterns. Two gels containing 38 lanes of SmaI-digested Enterococcus faecalis OG1RF DNA were analyzed to assess the impact of (i) varying the lane position of the standards, (ii) using gel plugs made at different times, and (iii) normalizing the fragment patterns by using molecular weight (MW) algorithms versus retardation factor (R(f)) algorithms. Two sets of PFGE libraries (one containing SmaI restriction patterns from 62 Enterococcus faecium isolates and the other containing SmaI restriction patterns of 89 Staphylococcus aureus isolates) were analyzed to assess the impact of varying the matching tolerance algorithm (designated as the vector box setting [VBS]) in the Phoretix software. Varying the lane position of standards on a gel and using gel plugs made on different days resulted in different VBSs, although it was not possible to judge whether those differences were statistically significant. Normalization of E. faecalis OG1RF fragment patterns by R(f) and MW methodology yielded no statistically significant differences in variability between the same fragment on different lanes. Suboptimal VBSs decreased the specificity with which related isolates were grouped together in dendrograms. The optimal VBS for analysis of PFGE fragment patterns from E. faecalis isolates differed from that for S. aureus isolates and sometimes was not that recommended by the manufacturer. Thus, computer-assisted analysis of PFGE patterns seemed to compensate for the intra- and intergel variation evaluated in the present study, and optimizing the software for the species to be tested was a critical preliminary step before further PFGE library analysis.     

Identification of clinical yeast isolates by using the Microring YT.

The Microring YT (MYT; Medical Wire & Equipment Co., Victory Gardens, N.J.) is a system for the rapid (24 to 48 h) identification of yeasts. The MYT system was evaluated and compared with the API20C (Analytab Products, Plainview, N.Y.) system for its ability to identify 677 clinical yeast isolates. Only 458 isolates (68%) were correctly identified by the MYT system, and the accuracy of the system varied considerably (0 to 96%), depending on the species. While MYT was less expensive and convenient to use and results were available 24 h sooner, it is inadequate for identification of many commonly isolated yeasts and is not designed for the identification of Cryptococcus species.     

In vitro activities of Daptomycin, Linezolid, and Quinupristin-Dalfopristin against a challenge panel of Staphylococci and Enterococci, including vancomycin-intermediate staphylococcus aureus and vancomycin-resistant Enterococcus faecium

We assessed the in vitro activities of daptomycin, linezolid, and quinupristin-dalfopristin (QD) against a contemporary challenge panel of 88 staphylococcal and 90 enterococcal isolates. The staphylococci selected included vancomycin-intermediate Staphylococcus aureus (VISA), methicillin-resistant S. aureus, and coagulasenegative staphylococci. Enterococcal isolates included vancomycin-resistant Enterococcus faecium (VREF) containing either vanA, vanB1, or vanD. The MICs of daptomycin, linezolid, and QD were determined using commercial broth microdilution panels. All three VISA isolates were susceptible to daptomycin, linezolid, and QD. QD was the most active agent against staphylococcal isolates (MIC50 < or = 0.5 microg/ml and MIC90 = 1 microg/ml), including those with decreased susceptibility to vancomycin. QD was also the most active agent against VREF (MIC90 < or = 0.5 microg/ml). No differences were seen for susceptibility of vanA, vanB1, and vanD VREF strains for daptomycin, linezolid, or QD. Daptomycin was the most effective against E. faecalis. On the basis of manufacturer-suggested interpretive criteria, 92% of isolates were susceptible (MIC90 = 4 microg/ml). All isolates tested were susceptible to at least one antimicrobial agent for which interpretive criteria have been defined. Population analysis of three S. aureus isolates for which the daptomycin MICs were 8 microg/ml showed a pattern of homogeneous resistance.