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Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions.

Viral replication places oncolytic viruses (OVs) in a unique niche in the field of drug pharmacokinetics (PK) as their self-amplification obscures exposure-response relationships. Moreover, standard bioanalytical techniques are unable to distinguish the input from replicated drug products. Here, we combine two novel approaches to characterize PK and biodistribution (BD) after systemic administration of vesicular stomatitis virus pseudotyped with lymphocytic choriomeningitis virus glycoprotein (VSV-GP) in healthy mice . First: to decouple input drug PK/BD versus replication PK/BD, we developed and fully characterized a replication-incompetent tool virus that retained all other critical attributes of the drug. We used this approach to quantify replication in blood and tissues and to determine its impact on PK and BD. Second: to discriminate the genomic and antigenomic viral RNA strands contributing to replication dynamics in tissues, we developed an in situ hybridization method using strand-specific probes and assessed their spatiotemporal distribution in tissues. This latter approach demonstrated that distribution, transcription, and replication localized to tissue-resident macrophages, indicating their role in PK and BD. Ultimately, our study results in a refined PK/BD profile for a replicating OV, new proposed PK parameters, and deeper understanding of OV PK/BD using unique approaches that could be applied to other replicating vectors., Competing Interests: The authors are/were employed at Boehringer Ingelheim Pharmaceuticals Inc. (BIPI) in Ridgefield, CT, at the time of the experiments., (© 2022.)

The foundation of pharmacokinetics and antidrug antibodies assay robustness relies on the use of high-quality reagents. Over the past decade, there has been increasing interest within the pharmaceutical industry, as well as regulators, on defining best practices and scientific approaches for generation, characterization and handling of critical reagents. In this review, we will discuss current knowledge and practices on critical reagent workflows and state-of-the-art approaches for characterization, generation, stability and storage and how each of these steps can impact ligand-binding assay robustness.

Irradiation of semiconductor surfaces often leads to the spontaneous formation of rippled structures at certain irradiation angles. However, at high enough energies, in the keV range, these structures are sometimes observed to vanish for all angles, despite the absence of any identified, universally-stabilizing physical mechanisms in operation. Here, we examine the effect on pattern formation of radiation-induced swelling, which has been excluded from prior treatments of stress in irradiated films. After developing a suitable continuum model, we perform a linear stability analysis to determine its effect on stability. Under appropriate simplifying assumptions, we find swelling indeed to be stabilizing at all angles for wavenumbers typical of experimental observations. Therefore, this mechanism may account for the suppression of ripple formation observed at energies over 1 keV.

Linear-regime Ar + bombardment of Si produces symmetrical ripple structures at ion incidence angles above 45° measured off-normal (Madi 2009 J. Phys.: Condens. Matter 21). In the nonlinear regime, new behaviors emerge. In this paper, we present experimental results of ion bombardment that continues into the nonlinear regime until pattern saturation at multiple ion incidence angles, showing the evolution of their grazing incidence small-angle x-ray scattering (GISAXS) spectra as well as atomic force microscopy topographs of the final, saturated structures. Asymmetric structures emerge parallel to the direction of the projected ion beam on the sample surface, constituting a height asymmetry not found in the linear regime. We then present simulations of surface height evolution under ion bombardment using a nonlinear partial differential equation developed by Pearson and Bradley (2015 J. Phys.: Condens. Matter 27 015010). We present simulated GISAXS spectra from these simulations, as well as simulated scattering from a sawtooth structure using the FitGISAXS software package (Babonneau 2010 J. Appl. Crystallogr. 43 929-36), and compare the simulated spectra to those observed experimentally. We find that these simulations reproduce many features of the sawtooth structures, as well as the nearly-flat final GISAXS spectra observed experimentally perpendicular to the sawtooth structures. However, the model fails to reproduce the final GISAXS spectra observed parallel to the sawtooth structures.

Metagenomic sequencing provides a window into microbial community structure and metabolic potential; however, linking these data to exogenous metabolites that microorganisms process and produce (the exometabolome) remains challenging. Previously, we observed strong exometabolite niche partitioning among bacterial isolates from biological soil crust (biocrust). Here we examine native biocrust to determine if these patterns are reproduced in the environment. Overall, most soil metabolites display the expected relationship (positive or negative correlation) with four dominant bacteria following a wetting event and across biocrust developmental stages. For metabolites that were previously found to be consumed by an isolate, 70% are negatively correlated with the abundance of the isolate's closest matching environmental relative in situ, whereas for released metabolites, 67% were positively correlated. Our results demonstrate that metabolite profiling, shotgun sequencing and exometabolomics may be successfully integrated to functionally link microbial community structure with environmental chemistry in biocrust.

Eukaryotic genomes are broadly divided between gene-rich euchromatin and the highly repetitive heterochromatin domain, which is enriched for proteins critical for genome stability and transcriptional silencing. This study shows that Drosophila KDM4A (dKDM4A), previously characterized as a euchromatic histone H3 K36 demethylase and transcriptional regulator, predominantly localizes to heterochromatin and regulates heterochromatin position-effect variegation (PEV), organization of repetitive DNAs, and DNA repair. We demonstrate that dKDM4A demethylase activity is dispensable for PEV. In contrast, dKDM4A enzymatic activity is required to relocate heterochromatic double-strand breaks outside the domain, as well as for organismal survival when DNA repair is compromised. Finally, DNA damage triggers dKDM4A-dependent changes in the levels of H3K56me3, suggesting that dKDM4A demethylates this heterochromatic mark to facilitate repair. We conclude that dKDM4A, in addition to its previously characterized role in euchromatin, utilizes both enzymatic and structural mechanisms to regulate heterochromatin organization and functions., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Mupirocin susceptibility testing of Staphylococcus aureus has become more important as mupirocin is used more widely to suppress or eliminate S. aureus colonization and prevent subsequent health care- and community-associated infections. The present multicenter study evaluated two susceptibility testing screening methods to detect mupirocin high-level resistance (HLR), broth microdilution (BMD) MICs of >or=512 microg/ml, and a 6-mm zone diameter for a disk diffusion (DD) test with a 200-microg disk. Initial testing indicated that with Clinical and Laboratory Standards Institute methods for BMD and DD testing, the optimal conditions for the detection of mupirocin HLR were 24 h of incubation and reading of the DD zone diameters with transmitted light. Using the presence or absence of mupA as the "gold standard" for HLR, the sensitivity and specificity of a single-well 256 microg/ml BMD test were 97 and 99%, respectively, and those for the 200-microg disk test were 98 and 99%, respectively. Testing with two disks, 200 microg and 5 microg, was evaluated for its ability to distinguish HLR isolates (MICs >or= 512 microg/ml), low-level-resistant (LLR) isolates (MICs = 8 to 256 microg/ml), and susceptible isolates (MICs

We compared the results obtained with six commercial MIC test systems (Etest, MicroScan, Phoenix, Sensititre, Vitek Legacy, and Vitek 2 systems) and three reference methods (agar dilution, disk diffusion, and vancomycin [VA] agar screen [VScr]) with the results obtained by the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference method for the detection of VA-intermediate Staphylococcus aureus (VISA). A total of 129 S. aureus isolates (VA MICs by previous BMD tests,

This report describes the results of an 11-laboratory study to determine if a cefoxitin broth microdilution MIC test could predict the presence of mecA in staphylococci. Using breakpoints of < or = 4 microg/ml for mecA-negative and > or = 6 or 8 microg/ml for mecA-positive isolates, sensitivity and specificity based on mecA or presumed mecA for Staphylococcus aureus at 18 h of incubation were 99.7 to 100% in three cation-adjusted Mueller-Hinton broths tested. For coagulase-negative strains at 24 h of incubation, breakpoints of < or = 2 microg/ml for mecA-negative and > or = 4 microg/ml for mecA-positive isolates gave sensitivity and specificity of 94 to 99% and 69 to 80%, respectively.

A study conducted by 11 laboratories investigated the ability of four combinations of erythromycin (ERY) and clindamycin (CC) (ERY and CC at 4 and 0.5, 6 and 1, 8 and 1.5, and 0.5 and 2 microg/ml) in a single well of a broth microdilution panel to predict the presence of inducible CC resistance. Each laboratory tested approximately 30 Staphylococcus aureus isolates and 20 coagulase-negative staphylococcus (CoNS) isolates in a panel using cation-adjusted Mueller-Hinton broth from three different manufacturers. Only the strains resistant to ERY and those susceptible or intermediate to CC were included in the analysis (S. aureus, n = 333; CoNS, n = 97). Results of the D-zone test were used as the gold standard. After an 18-h incubation, the combination of 4 microg/ml ERY and 0.5 microg/ml CC performed the best, with 98 to 100% sensitivity and 100% specificity for both organism groups. After a 24-h incubation, the ERY-CC combinations of 4 and 0.5, 6 and 1, and 8 and 1.5 microg/ml correlated well with the D-zone test.

Phenotypic methods for detecting mecA-mediated resistance in Staphylococcus aureus include both oxacillin and cefoxitin susceptibility tests; many laboratories perform multiple tests. Conflicting oxacillin and cefoxitin susceptibility results are most likely to occur for isolates that either have reduced susceptibility to oxacillin by a non-mecA-mediated mechanism or are mecA positive but are very heteroresistant. To understand the performance of oxacillin and cefoxitin tests for such isolates, we tested 135 S. aureus isolates using either cefoxitin or oxacillin and compared the results with mecA polymerase chain reaction. These strains either expressed borderline oxacillin MICs (1-4 microg/mL) and had undetermined mecA status or were mecA positive but were not detected by oxacillin broth microdilution (BMD) or disk diffusion (DD) in original testing. For 24-h readings, performance of cefoxitin tests (sensitivity/specificity) were DD (99/100), Etest using < or =6 microg/mL as susceptible (99/98), and Phoenix MIC using < or =4 microg/mL as susceptible (98/100). Using 6 microg/mL of cefoxitin as a screen test in both BMD and agar dilution also worked well (98/98-100). Sensitivity/specificity of oxacillin methods were oxacillin agar screen (BBL: 80/86; Remel, Lenexa, KS: 85/50), DD (91/59), BMD (85/88), MicroScan (89/96), VITEK Legacy (82/93), VITEK 2 (91/73), and Phoenix, (67/96). These results suggest that a cefoxitin test can be used alone to predict mecA-mediated resistance in S. aureus.

We reevaluated Enterobacteriaceae disk diffusion breakpoints for the tetracyclines published in the Clinical and Laboratory Standards Institute (CLSI) document M100-S16, which were (susceptible/resistant) >or=19 mm/or=16 mm/or=19 mm/or=15 mm/or=14 mm/or=16 mm/

Acinetobacter sp. isolates having multidrug resistance (MDR) patterns have become common in many medical centers worldwide, limiting therapeutic options. A five-center study tested 103 contemporary clinical Acinetobacter spp., including MDR strains, by reference broth microdilution and disk diffusion (15-mug disk content) methods against tigecycline. Applying U.S. Food and Drug Administration tigecycline breakpoint criteria for Enterobacteriaceae (susceptibility at < or =2 microg/ml [< or =1 microg/ml by the European Committee on Antimicrobial Susceptibility Testing]; disk diffusion breakpoints at > or =19 mm and < or =14 mm) to Acinetobacter spp. led to an unacceptable error rate (23.3%). However, an adjustment of tigecycline disk diffusion breakpoints (susceptible/resistant) to > or =16/ < or =12 mm reduced intermethod errors to an acceptable level (only 9.7%, all minor).

The cefoxitin disk diffusion (DD) test for predicting mecA-mediated oxacillin resistance in staphylococci was assessed during a three-phase study. In phase 1, one laboratory tested 62 and 53 strains of Staphylococcus aureus and coagulase-negative staphylococci (CoNS), respectively. These data were used to choose the provisional cefoxitin DD breakpoints (resistant/susceptible) of < or =19 mm/> or =20 mm for S. aureus and < or =24 mm/> or =25 mm for CoNS for the next phase of testing. In phase 2, 10 laboratories each tested approximately 40 in-house strains of staphylococci (half of which were S. aureus) using Mueller-Hinton agar from different manufacturers. In this phase, the sensitivity and specificity, respectively, of the cefoxitin disk test were 98 and 100% for S. aureus and 99 and 96% for CoNS. The cefoxitin DD test performed equivalently to oxacillin broth microdilution (BMD) and to oxacillin DD tests among S. aureus and mecA-positive CoNS strains but gave better results than oxacillin BMD or oxacillin DD for mecA-negative strains of CoNS. The cefoxitin DD test also was much easier to read and did not require the use of transmitted light for detection of resistance. Based on data from the first two phases, the Clinical and Laboratory Standards Institute (CLSI; formerly NCCLS) adopted the use of the cefoxitin DD test for predicting mecA-mediated oxacillin resistance in staphylococci and revised Table 2C in CLSI document M100-S14 to reflect the change. In the third phase, an additional 61 challenge strains of CoNS for which the oxacillin MICs were 0.5 to 2 microg/ml were tested in a single laboratory to determine the effectiveness of the cefoxitin DD test for this group of borderline-resistant isolates. These data were used to refine the description of the test in CLSI document M100-S15. The cefoxitin DD test is preferred over the oxacillin DD test for predicting mecA-mediated oxacillin resistance in S. aureus and CoNS.

An eight-laboratory study addressed the urgent need for quality control (QC) ranges for susceptibility determination when testing colistin (polymyxin E) and polymyxin B, two polycationic peptide antimicrobial agents, against multidrug-resistant gram-negative bacilli. For Escherichia coli ATCC 25922l, the QC ranges were as follows: for colistin, 0.25 to 1 microg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 microg/ml (13 to 19 mm). For Pseudomonas aeruginosa ATCC 27853, the QC ranges were as follows: for colistin, 0.25 to 2 microg/ml (11 to 17 mm), and for polymyxin B, 0.25 to 2 mug/ml (14 to 18 mm). More than 97% of all reported QC results were within these proposed ranges.

Although both broth microdilution (BMD) and disk diffusion (DD) are listed by NCCLS as acceptable methods for testing Acinetobacter spp. for antimicrobial susceptibility, few studies have compared the results generated by the two methods. We tested 196 isolates of Acinetobacter spp. from nine U.S. hospitals and from the Centers for Disease Control culture collection by using BMD and DD and clinically appropriate antimicrobial agents. Categorical results for amikacin, ciprofloxacin, gatifloxacin, gentamicin, imipenem, levofloxacin, meropenem, tobramycin, and trimethoprim-sulfamethoxazole were comparable for the two methods: there was only one very major (VM) error, with tobramycin, and only one major (M) error, with meropenem, when DD results were compared with BMD results. However, VM errors were frequent with the beta-lactams and beta-lactam-beta-lactam inhibitor combinations, while M errors were often observed with tetracyclines. For BMD, tests frequently exhibited subtle growth patterns that were difficult to interpret, especially for beta-lactams. If subtle growth (i.e., granular, small button, or "starry" growth) was considered positive, error rates between BMD and DD were unacceptably high for ampicillin-sulbactam (VM error, 9.8%; minor [m] error, 16.1%), piperacillin (VM error, 5.7%; m error, 13.5%), piperacillin-tazobactam (VM error, 9.3%; m error, 12.9%), ceftazidime (VM error, 6.2%; m error, 11.4%), cefepime (VM error, 6.2%; m error, 13.0%), cefotaxime (m error, 21.2%), ceftriaxone (m error, 23.3%), tetracycline (M error, 11.4%; m error, 32.1%), and doxycycline (M error, 2.6%). When subtle growth patterns were ignored, the agreement still did not achieve acceptable levels. To determine if the problems with BMD testing occurred in other laboratories, we sent frozen BMD panels containing beta-lactam drugs and nine isolates to six labs with experience in performing BMD and DD. Among these laboratories, cefepime MICs ranged from < or =8 to > or =32 microg/ml for four of the nine strains, confirming the problem in interpreting BMD results. Discrepancies between the categorical interpretations of BMD and DD tests were noted primarily with cefepime and piperacillin, for which the BMD results were typically more resistant. Clinical laboratories should be aware of these discrepancies. At present, there are no data to indicate which method provides more clinically relevant information.

From January 1996 to May 1999, Project ICARE (Intensive Care Antimicrobial Resistance Epidemiology) received 448 nonduplicate clinical isolates of Enterobacteriaceae and Pseudomonas aeruginosa that were reported to be imipenem intermediate or resistant. However, broth microdilution (BMD) confirmatory testing at the Project ICARE central laboratory confirmed this result in only 11 of 123 (8.9%) Enterobacteriaceae isolates and 241 of 325 (74.2%) P. aeruginosa isolates. To investigate this overdetection of imipenem resistance, we tested 204 selected isolates from the Project ICARE collection plus five imipenem-resistant challenge strains at the Centers for Disease Control and Prevention against imipenem and meropenem by agar dilution, disk diffusion, Etest (AB BIODISK North America, Inc., Piscataway, N.J.), two MicroScan WalkAway conventional panels (Neg MIC Plus 3 and Neg Urine Combo 3) (Dade MicroScan, Inc., West Sacramento, Calif.), and two Vitek cards (GNS-116 containing meropenem and GNS-F7 containing imipenem) (bioMérieux Vitek, Inc., Durham, N.C.). The results of each test method were compared to the results of BMD testing using in-house-prepared panels. Seven imipenem-resistant and five meropenem-resistant isolates of Enterobacteriaceae and 43 imipenem-resistant and 21 meropenem-resistant isolates of P. aeruginosa were identified by BMD. For Enterobacteriaceae, the imipenem and meropenem test methods produced low numbers of very major and major errors. All test systems in the study produced low numbers of very major and major errors when P. aeruginosa was tested against imipenem and meropenem, except for Vitek testing (major error rate for imipenem, 20%). Further testing conducted in 11 of the participating ICARE hospital laboratories failed to pinpoint the factors responsible for the initial overdetection of imipenem resistance. However, this study demonstrated that carbapenem testing difficulties do exist and that laboratories should consider using a second, independent antimicrobial susceptibility testing method to validate carbapenem-intermediate and -resistant results.

The need for new antimicrobial agents with activity against Gram-positive organisms has become increasingly important because of emerging resistance. We compared the activity of a new b-lactam antimicrobial agent, RWJ-54428 (MC-02 479), with representatives of other classes of antimicrobial agents against 76 Staphylococcus aureus (including four glycopeptide- intermediate strains), 50 coagulase-negative staphylococci, 20 Enterococcus faecalis, 20 Enterococcus faecium, 10 Enterococcus gallinarum/Enterococcus casseliflavus, 54 Streptococcus pneumoniae and 22 viridans streptococcal isolates. The MIC(90) of RWJ-54,428 was < or = 2 mg/L for all groups of bacteria tested except E. faecium. The activity against four strains of glycopeptide-intermediate S. aureus was similar to that for other methicillin-resistant S. aureus isolates (range 0.5-2.0 mg/L).

To define more precisely the inoculation methods to be used in the oxacillin screen test for Staphylococcus aureus, we tested agar screen plates prepared in house with 6 microg of oxacillin/ml and 4% NaCl using the four different inoculation methods that would most likely be used by clinical laboratories. The organisms selected for testing were 19 heteroresistant mecA-producing strains and 41 non-mecA-producing strains for which oxacillin MICs were near the susceptible breakpoint. The inoculation method that was preferred by all four readers and that resulted in the best combination of sensitivity and specificity was a 1-microl loopful of a 0.5 McFarland suspension. A second objective of the study was to then use this method to inoculate plates from five different manufacturers of commercially prepared media. Although all commercial media performed with acceptable sensitivity compared to the reference lot, one of the commercial lots demonstrated a lack of specificity. Those lots of oxacillin screen medium that fail to grow heteroresistant strains can be detected by using S. aureus ATCC 43300 as a positive control in the test and by using transmitted light to carefully examine the plates for any growth. However, lack of specificity with commercial lots may be difficult to detect using any of the current quality control organisms.

Using a set of 55 Staphylococcus aureus challenge organisms, we evaluated six routine methods (broth microdilution, disk diffusion, oxacillin agar screen, MicroScan conventional panels, MicroScan rapid panels, and Vitek cards) currently used in many clinical laboratories and two new rapid methods, Velogene and the MRSA-Screen, that require less than a day to determine the susceptibility of S. aureus to oxacillin. The methods were evaluated by using the presence of the mecA gene, as detected by PCR, as the "gold standard." The strains included 19 mecA-positive heterogeneously resistant strains of expression class 1 or 2 (demonstrating oxacillin MICs of 4 to >16 microg/ml) and 36 mecA-negative strains. The oxacillin MICs of the latter strains were 0.25 to 4 microg/ml when tested by broth microdilution with 2% NaCl-supplemented cation-adjusted Mueller-Hinton broth as specified by the NCCLS. However, when tested by agar dilution with 4% salt (the conditions used in the oxacillin agar screen method), the oxacillin MICs of 16 of the mecA-negative strains increased to 4 to 8 microg/ml. On initial testing, the percentages of correct results (% sensitivity/% specificity) were as follows: broth microdilution, 100/100; Velogene, 100/100; Vitek, 95/97; oxacillin agar screen, 90/92; disk diffusion, 100/89; MicroScan rapid panels, 90/86; MRSA-Screen, 90/100; and MicroScan conventional, 74/97. The MRSA-Screen sensitivity improved to 100% if agglutination reactions were read at 15 min. Repeat testing improved the performance of some but not all of the systems.

The activities of two investigational fluoroquinolones and three fluoroquinolones that are currently marketed were determined for 182 clinical isolates of Streptococcus pneumoniae. The collection included 57 pneumococcal isolates resistant to levofloxacin (MIC >/= 8 microg/ml) recovered from patients in North America and Europe. All isolates were tested with clinafloxacin, gatifloxacin, gemifloxacin, levofloxacin, and trovafloxacin by the National Committee for Clinical Laboratory Standards broth microdilution and disk diffusion susceptibility test methods. Gemifloxacin demonstrated the greatest activity on a per gram basis, followed by clinafloxacin, trovafloxacin, gatifloxacin, and levofloxacin. Scatterplots of the MICs and disk diffusion zone sizes revealed a well-defined separation of levofloxacin-resistant and -susceptible strains when the isolates were tested against clinafloxacin and gatifloxacin. DNA sequence analyses of the quinolone resistance-determining regions of gyrA, gyrB, parC, and parE from 21 of the levofloxacin-resistant strains identified eight different patterns of amino acid changes. Mutations among the four loci had the least effect on the MICs of gemifloxacin and clinafloxacin, while the MICs of gatifloxacin and trovafloxacin increased by up to six doubling dilutions. These data indicate that the newer fluoroquinolones have greater activities than levofloxacin against pneumococci with mutations in the DNA gyrase or topoisomerase IV genes. Depending upon pharmacokinetics and safety, the greater potency of these agents could provide improved clinical efficacy against levofloxacin-resistant pneumococcal strains.

Klebsiella pneumoniae K6 (ATCC 700603), a clinical isolate, is resistant to ceftazidime and other oxyimino-beta-lactams. A consistent reduction in the MICs of oxyimino-beta-lactams by at least 3 twofold dilutions in the presence of clavulanic acid confirmed the utility of K. pneumoniae K6 as a quality control strain for extended-spectrum beta-lactamase (ESBL) detection. Isoelectric-focusing analysis of crude lysates of K6 demonstrated a single beta-lactamase with a pI of 7.8 and a substrate profile showing preferential hydrolysis of cefotaxime compared to ceftazidime. PCR analysis of total bacterial DNA from K6 identified the presence of a bla(SHV) gene. K6 contained two large plasmids with molecular sizes of approximately 160 and 80 kb. Hybridization of plasmid DNA with a bla(SHV)-specific probe indicated that a bla(SHV) gene was encoded on the 80-kb plasmid, which was shown to transfer resistance to ceftazidime in conjugal mating experiments with Escherichia coli HB101. DNA sequencing of this bla(SHV)-related gene revealed that it differs from bla(SHV-1) at nine nucleotides, five of which resulted in amino acid substitutions: Ile to Phe at position 8, Arg to Ser at position 43, Gly to Ala at position 238, and Glu to Lys at position 240. In addition to the production of this novel ESBL, designated SHV-18, analysis of the outer membrane proteins of K6 revealed the loss of the OmpK35 and OmpK37 porins.

A multilaboratory study was undertaken to determine the accuracy of the current National Committee for Clinical Laboratory Standards (NCCLS) oxacillin breakpoints for broth microdilution and disk diffusion testing of coagulase-negative staphylococci (CoNS) by using a PCR assay for mecA as the reference method. Fifty well-characterized strains of CoNS were tested for oxacillin susceptibility by the NCCLS broth microdilution and disk diffusion procedures in 11 laboratories. In addition, organisms were inoculated onto a pair of commercially prepared oxacillin agar screen plates containing 6 microg of oxacillin per ml and 4% NaCl. The results of this study and of several other published reports suggest that, in order to reliably detect the presence of resistance mediated by mecA, the oxacillin MIC breakpoint for defining resistance in CoNS should be lowered from >/=4 to >/=0.5 microg/ml and the breakpoint for susceptibility should be lowered from /=18 mm for susceptibility is suggested. Due to the poor sensitivity of the oxacillin agar screen plate for predicting resistance in this study, this test can no longer be recommended for use with CoNS. The proposed interpretive criteria for testing CoNS have been adopted by the NCCLS.

Background: Transplant coronary arteriopathy causes late death and is difficult to detect noninvasively. Dobutamine stress echocardiography is being used for risk stratification in adult recipients at some transplant centers, thus we investigated its role in a pediatric population., Methods: We performed 46 stress echo studies (mean age = 11.8 years; mean years post transplantation = 4.3). An atropine/dobutamine protocol (5-40 mcg/kg/min) was used to attain a predicted target heart rate. Serial echocardiographic images were acquired at baseline and at each increment of dobutamine and recovery, and were digitized online. Data were correlated with endomyocardial biopsy (n = 23), coronary angiography (n = 26) or autopsy (n = 6). All studies were well tolerated., Results: Target heart rate was achieved in 41/46 (89%) studies. The mean heart rate significantly increased from 95 to 169 beats/min and mean systolic blood pressure from 123 to 153 mm Hg (p<.05). The mean peak pressure-rate product was 23,041 beats-mm Hg/min. Coronary arteriopathy was confirmed in 5 patients by angiography (n = 3) explanted heart (n = 1) or autopsy (n = 4). In this group, abnormalities included a new reversible wall motion abnormality (n = 2), left ventricular cavity dilation with stress (n = 3), ischemia (n = 2), increased mitral insufficiency (n = 1) and marked diastolic dysfunction (n = 1). A positive study predicted death or graft failure (p< .0005)., Conclusions: Echocardiographic abnormalities during stress correlated with coronary arteriopathy in this small cohort of patients; however, larger multi-center studies are warranted to assess the utility of dobutamine stress echocardiography for risk stratification for coronary disease in pediatric transplant recipients.

Fluoroquinolone resistance appears to be increasing in many species of bacteria, particularly in those causing nosocomial infections. However, the accuracy of some antimicrobial susceptibility testing methods for detecting fluoroquinolone resistance remains uncertain. Therefore, we compared the accuracy of the results of agar dilution, disk diffusion, MicroScan Walk Away Neg Combo 15 conventional panels, and Vitek GNS-F7 cards to the accuracy of the results of the broth microdilution reference method for detection of ciprofloxacin and ofloxacin resistance in 195 clinical isolates of the family Enterobacteriaceae collected from six U.S. hospitals for a national surveillance project (Project ICARE [Intensive Care Antimicrobial Resistance Epidemiology]). For ciprofloxacin, very major error rates were 0% (disk diffusion and MicroScan), 0.9% (agar dilution), and 2.7% (Vitek), while major error rates ranged from 0% (agar dilution) to 3.7% (MicroScan and Vitek). Minor error rates ranged from 12.3% (agar dilution) to 20.5% (MicroScan). For ofloxacin, no very major errors were observed, and major errors were noted only with MicroScan (3.7% major error rate). Minor error rates ranged from 8.2% (agar dilution) to 18.5% (Vitek). Minor errors for all methods were substantially reduced when results with MICs within +/-1 dilution of the broth microdilution reference MIC were excluded from analysis. However, the high number of minor errors by all test systems remains a concern.

Resistance to fluoroquinolone (FQ) antibiotics in Streptococcus pneumoniae has been attributed primarily to specific mutations in the genes for DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE). Resistance to some FQs can result from a single mutation in one or more of the genes encoding these essential enzymes. A group of 160 clinical isolates of pneumococci was examined in this study, including 36 ofloxacin-resistant isolates (MICs, > or = 8 micrograms/ml) recovered from patients in North America, France, and Belgium. The susceptibilities of all isolates to clinafloxacin, grepafloxacin, levofloxacin, sparfloxacin, and trovafloxacin were examined by the National Committee for Clinical Laboratory Standards reference broth microdilution and disk diffusion susceptibility testing methods. Among the ofloxacin-resistant strains, 32 of 36 were also categorized as resistant to levofloxacin, 35 were resistant to sparfloxacin, 29 were resistant to grepafloxacin, and 19 were resistant to trovafloxacin. In vitro susceptibility to clinafloxacin appeared to be least affected by resistance to the other FQs. Eight isolates with high- and low-level resistance to the newer FQs were selected for DNA sequence analysis of the quinolone resistance-determining regions (QRDRs) of gyrA, gyrB, parC, and parE. The DNA and the inferred amino acid sequences of the resistant strains were compared with the analogous sequences of reference strain S. pneumoniae ATCC 49619 and FQ-susceptible laboratory strain R6. Reduced susceptibilities to grepafloxacin and sparfloxacin (MICs, 1 to 2 micrograms/ml) and trovafloxacin (MICs, 0.5 to 1 microgram/ml) were associated with either a mutation in parC that led to a single amino acid substitution (Ser-79 to Phe or Tyr) or double mutations that involved the genes for both GyrA (Ser-81 to Phe) and ParE (Asp-435 to Asn). High-level resistance to all of the compounds except clinafloxacin was associated with two or more amino acid substitutions involving both GyrA (Ser-81 to Phe) and ParC (Ser-79 to Phe or Ser-80 to Pro and Asp-83 to Tyr). No mutations were observed in the gyrB sequences of resistant strains. These data indicate that mutations in pneumococcal gyrA, parC, and parE genes all contribute to decreased susceptibility to the newer FQs, and genetic analysis of the QRDR of a single gene, either gyrA or parC, is not predictive of pneumococcal resistance to these agents.

Genes encoding streptomycin/spectinomycin adenylyltransferases [ANT(3")(9)] have been reported to exist in gram-negative organisms and Staphylococcus aureus. During a study of high-level aminoglycoside resistance in enterococci, we encountered an isolate of Enterococcus faecalis that was streptomycin resistant but did not appear to contain the 6'-adenylyltransferase gene (aadE) when examined by PCR with specific primers. Phosphocellulose paper binding assays indicated the presence of an ANT(3")(9) enzyme. Streptomycin and spectinomycin MICs of 4,000 and 8,000 microg/ml, respectively, were observed for the isolate. PCR primers corresponding to a highly conserved region of the aadA gene were used to amplify a specific 284-bp product. The product hybridized with a digoxigenin-labeled PCR product from E. coli C600(pHP45Omega) known to contain the aadA gene. The aadA gene was transferred via filter matings from the E. faecalis donor to E. faecalis JH2-2. PCR primers designed for analysis of integrons were used to amplify a 1-kb product containing the aadA gene, which was cloned into the vector pCRII and transformed into Escherichia coli DH5-alpha competent cells. D-Rhodamine dye terminator cycle sequencing was used to determine the gene sequence, which was compared to previously reported sequences of aadA genes. We found the aadA gene in E. faecalis to be identical to the aadA genes reported by Sundstr om et al. for E. coli plasmid R6-5 (L. Sundström, P. Râdström, G. Swedberg, and O. Sköld, Mol. Gen. Genet. 213:191-201, 1988), by Fling et al. for the aadA within transposon Tn7 (M. E. Fling, J. Kopf, and C. Richards, Nucleic Acids Res. 13:7095-7106, 1985), and by Hollingshead and Vapnek for E. coli R538-1 (S. Hollingshead and D. Vapnek, Plasmid 13:17-30, 1985). Previous reports of the presence of the aadA gene in enterococci appear to be erroneous and probably describe an aadE gene, since the isolates were reported to be susceptible to spectinomycin.

Few reports of vancomycin-resistant enterococci have appeared outside the USA. Therefore, we evaluated the ability of five laboratories in Buenos Aires, Argentina, to perform susceptibility testing using the disk diffusion method. Laboratories had difficulty identifying the low- and intermediate-level vancomycin-resistant phenotypes. This suggests that the disk diffusion method used by laboratories abroad may fail to detect some vancomycin-resistant enterococci.

A six-laboratory collaborative study was conducted to develop MIC and zone diameter quality control limits and interpretive criteria for antimicrobial susceptibility testing of Streptococcus pneumoniae with azithromycin, clarithromycin, dirithromycin, and clindamycin. The MICs of all of the agents plus erythromycin for 302 clinical isolates of pneumococci that had been selected with an emphasis on resistant strains were determined by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure. The zone diameters of the isolates were also determined for the same agents except erythromycin by the NCCLS disk diffusion test procedure. Repeated testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of MIC and zone diameter quality control ranges for these agents. Interpretive criteria for the MIC of azithromycin were established and were as follows: susceptible, < or = 0.5 microgram/ml; intermediate, 1 microgram/ml; and resistant, > or = 2 micrograms/ml. The interpretive criteria advocated for the MICs of clarithromycin and clindamycin were as follows: susceptible, < or = 0.25 microgram/ml; intermediate, 0.5 microgram/ml; and resistant, > or = 1 microgram/ml. Comparison of MICs and disk diffusion zone diameters led to the development of interpretive criteria for the zone diameters for azithromycin, clarithromycin, and clindamycin that correlated well with these MIC breakpoints. Testing of this organism collection also led to the reestablishment of the erythromycin MIC breakpoints as being identical to those of clarithromycin, which resulted in equivalent cross-susceptibility and cross-resistance for the three macrolides that are currently marketed in the United States. Thus, the susceptibility of pneumococci to azithromycin and clarithromycin can be predicted accurately by testing only erythromycin in clinical laboratories. This recommendation, as well as the interpretive and quality control criteria that are described, have been accepted by NCCLS and are included in the latest NCCLS susceptibility testing guidelines.

Seven commercial systems for antimicrobial susceptibility testing of Streptococcus pneumoniae were evaluated by using a challenge set of 55 pneumococcal isolates with a variety of resistance phenotypes and genotypes. Overall, the results produced by the Pasco and Etest methods were found to be acceptable for all drugs tested except for trimethoprim-sulfamethoxazole testing by the Etest. The Just One system for penicillin MIC testing was also judged to be acceptable (minor error rate, 5.5%). Although the Sensititre and MicroTech methods both produced 12.7% minor errors with penicillin, the Sensititre method classified penicillin-intermediate strains as resistant or vice versa, while four of MicroTech's errors were among intermediate strains that were classified as susceptible. The MicroMedia (minor error rate, 16.4%) and MicroScan Rapid (minor error rate, 63.6%) methods produced unacceptably high levels of errors when testing penicillin. Minor error rates for cefotaxime and ceftriaxone ranged from a low of 12.7% (Etest and Sensititre) to a high of 28% (MicroMedia). Error rates were low for erythromycin, tetracycline, and chloramphenicol by most methods with the exception of the MicroScan method, which had a high very major error rate for erythromycin (34.6%). For testing of beta-lactam drugs, the Pasco, Etest, and Just One tests for penicillin are the most accurate methods; the Sensititre method also provided acceptable results.

Since the early 1970s, the synergistic activity of an aminoglycoside with a cell wall-active agent has been predicted by determining the ability of an enterococcus to grow in the presence of high levels of the aminoglycoside (usually > or = 2,000 micrograms/ml). However, a variety of media and concentrations of aminoglycosides has been used for this screening procedure. In the present study, we sought to optimize the agar dilution, broth microdilution, and disk diffusion tests used to detect high-level gentamicin and streptomycin resistance in enterococci. For dilution tests, brain heart infusion agar or broth gave the best growth and performance. For agar dilution, 500 micrograms of gentamicin per ml, 2,000 micrograms of streptomycin per ml, and an inoculum of 1 x 10(6) CFU/ml were optimal, while for broth microdilution, 500 micrograms of gentamicin per ml, 1,000 micrograms of streptomycin per ml, and an inoculum of 5 x 10(5) CFU/ml were best. Growth of more than one colony in the agar dilution test was determined to be the best indicator of high-level resistance. For disk diffusion, Mueller-Hinton agar, 120-micrograms gentamicin disks, and 300-micrograms streptomycin disks with breakpoints of no zone for resistance and > or = 10 mm for susceptibility gave the best sensitivity and specificity if results for strains with zones of 7 to 9 mm are considered inconclusive, indicating that a broth or agar test should be performed to determine susceptibility or resistance.

Studies were conducted to validate the use of Enterococcus faecalis ATCC 51299 (which is vancomycin resistant and resistant to high levels of gentamicin and streptomycin) and E. faecalis ATCC 29212 (which is susceptible to vancomycin and against which gentamicin or streptomycin and cell wall-active agents have synergistic kill activity) as controls in an agar screening test for vancomycin resistance and high-level streptomycin and gentamicin resistance and a broth microdilution screening test for high-level streptomycin and gentamicin resistance. Both organisms performed as expected in these tests and will serve as appropriate controls. However, E. faecalis ATCC 29212 was occasionally noted to produce light growth on the vancomycin screening plate with certain lots of agar. Quality control ranges for disk diffusion tests with disks with large amounts of streptomycin (300 micrograms) and gentamicin (120 micrograms) were established for E. faecalis ATCC 29212; zone limits are 16 to 22 mm for gentamicin and 14 to 19 mm for streptomycin. No zones for inhibition were seen when E. faecalis ATCC 51299 was tested with these high-content disks.

We evaluated the abilities of 10 commercially available antimicrobial susceptibility testing methods and four reference methods (agar dilution, broth microdilution, disk diffusion, and the agar screen plate) to classify enterococci correctly as vancomycin susceptible or resistant using 50 well-characterized strains of enterococci. There was a high level of agreement of category classification data obtained with broth-based systems (Sceptor, MicroMedia, Pasco, and Sensititre), agar dilution, and an antibiotic gradient method (E test) with data obtained by reference broth microdilution; no very major or major errors were seen, and minor errors were < or = 6%. Increased minor error rates were observed with disk diffusion (12%), Alamar (16%), Uniscept (16%), and conventional (overnight) MicroScan panels (16%). The errors were primarily with Enterococcus casseliflavus strains and organisms containing the vanB vancomycin resistance gene. Very major error rates of 10.3 and 20.7% were observed with Vitek and MicroScan Rapid (MS/Rapid) systems, respectively; however, only the MS/Rapid system produced major errors (13.3%). On repeat testing of discrepant isolates, the very major error rate with the Vitek system dropped to 3.4%, while the very major error rate with the MS/Rapid system increased to 27.6%; major errors with the MS/Rapid system were not resolved. Many of the commercial systems had only 4 dilutions of vancomycin, which resulted in up to 84% of values being off scale (e.g., Uniscept). Of the methods tested, most conventional broth- and agar-based methods proved to be highly accurate when incubation was done for a full 24 h, although several of the tests had high minor error rates. Automated systems continued to demonstrate problems in detecting low-level resistance.

Objectives: We studied rejection, allograft function and side effects, such as hypertension, renal dysfunction and hypercholesterolemia, in seven patients switched from cyclosporine-based triple-drug immunosuppression to FK 506., Background: A subset of pediatric heart transplant recipients treated with triple-drug immunosuppression consisting of cyclosporine, azathioprine and prednisone experience either persistent rejection when attempts are made to taper corticosteroids or morbidity from cyclosporine and corticosteroids. Experience with the new immunosuppressive agent FK 506 has demonstrated its effectiveness as a single agent in heart transplant recipients, and anecdotal evidence has shown that side effects such as hypertension and hypercholesterolemia may be lower., Methods: Seven patients whom we deemed corticosteroid dependent were switched to FK 506-based therapy. Allograft function, episodes of rejection, need for corticosteroids and incidence of side effects from FK 506 were monitored. The switch to FK 506 was performed using an established protocol. Follow-up time has ranged from 15 to 41 months. Serial right heart catheterizations and endomyocardial biopsies were performed after each reduction of corticosteroid dosing., Results: Catheterization data showed no significant change in pulmonary wedge pressure, mean right atrial pressure or cardiac index, indicating no decline in allograft function. Serial echocardiographic variables of allograft function were also stable. At present, all seven patients are free of the corticosteroid portion of their immune suppression. There have been only two episodes of significant acute rejection requiring treatment with intravenous corticosteroids. Antihypertensive medications have been discontinued in five of six patients previously treated with these drugs. Plasma cholesterol, low density lipoprotein and triglyceride levels were decreased, and renal function was stable., Conclusions: Preliminary studies suggest that FK 506 may be an alternative immunosuppressive agent for pediatric and adolescent patients experiencing ongoing rejection or significant morbidity from cyclosporine and corticosteroids and in those patients dependent on corticosteroids for immune suppression.

A five-center collaborative study was undertaken to develop quality control and specific interpretive criteria for susceptibility testing of Streptococcus pneumoniae against 12 antimicrobial agents. MICs were determined for 248 pneumococcal clinical isolates (with an emphasis on resistant strains) by use of the National Committee for Clinical Laboratory Standards (NCCLS)-recommended broth microdilution procedure incorporating lysed horse blood-supplemented Mueller-Hinton broth. NCCLS disk diffusion testing was also performed for each isolate by using Mueller-Hinton sheep blood agar incubated in 5% CO2. Repetitive testing of S. pneumoniae ATCC 49619 with different sources and lots of media and disks allowed development of quality control ranges which encompassed approximately 95% of MIC and zone size values observed in the study. Good intra- and interlaboratory reproducibilities were seen with these testing methods and all of the drugs examined. On the basis of the results of this study, MIC interpretive criteria are proposed for 11 agents. Comparisons of MICs and disk diffusion zone sizes allowed disk diffusion zone size interpretive criteria to be proposed for five drugs and confirmed the use of the oxacillin disk test for prediction of penicillin susceptibility among pneumococci. Excessive numbers of minor-category interpretive errors precludes recommendation at this time of the disk diffusion method for testing of pneumococci against five of the drugs. Use of these proposed quality control and interpretive criteria should provide for reproducible test results and allow recognition of recently emerging resistance among pneumococcal clinical isolates.

The incidence of vancomycin resistance among enterococci is increasing in the United States and elsewhere in the world, but automated susceptibility testing methods have difficulty detecting resistance expressed by certain strains. The agar screening method described by Willey et al. (B. M. Willey, B. N. Kreiswirth, A. E. Simor, G. Williams, S. R. Scriver, A. Phillips, and D. E. Low, J. Clin. Microbiol. 30:1621-1624, 1992) has been proposed as a reliable method for confirming vancomycin resistance. In this study, we investigated various parameters associated with the agar screening method and, on the basis of the findings, established optimum testing conditions for the method. First, to evaluate media and vancomycin concentrations, one laboratory used Mueller-Hinton and brain heart infusion agars supplemented with 4, 6, and 8 micrograms of vancomycin per ml to test 100 genetically characterized enterococcal strains. On the basis of the results obtained, brain heart infusion agar supplemented with 6 micrograms of vancomycin per ml was selected for further study. Subsequently, eight laboratories used the medium to test both reference and clinical isolates. There was very good performance with the reference strains and, among 158 clinical isolates tested, the method demonstrated sensitivity and specificity of 100% and from 96 to 99%, respectively.

Increasing penicillin resistance and the initial recognition of resistance to extended-spectrum cephalosporins among Streptococcus pneumoniae isolates have placed greater emphasis on accurate methods for susceptibility testing of clinical isolates. This study has evaluated the use of the E test (AB Biodisk NA, Piscataway, N.J.) for the detection of penicillin and cefotaxime resistance among 147 pneumococcal clinical isolates in three geographically separate laboratories. These included 42 penicillin-resistant (MIC, > or = 2 micrograms/ml) and 14 cefotaxime-resistant (defined here as an MIC of > or = 2 micrograms/ml) isolates. E test strips were applied to the surface of Mueller-Hinton sheep blood agar plates and incubated at 35 degrees C in 5% CO2 for 20 to 24 h. E test MICs were compared with MICs determined with lysed horse blood-supplemented Mueller-Hinton broth in a microdilution format as recommended by the National Committee for Clinical Laboratory Standards. Penicillin MICs agreed within one log2 dilution for 136 of 147 (92.5%) isolates, and cefotaxime MICs agreed within one log2 dilution for 142 of 147 (96.6%) isolates. No very major or major interpretive errors occurred with either penicillin or cefotaxime E test MIC results. There were 9.5 and 5.4% minor interpretive category errors with penicillin and cefotaxime E test MICs, respectively. These data indicate that the E test represents a convenient and reliable method for the detection of penicillin or cephalosporin resistance in pneumococci.

We examined 105 clinical isolates of glycopeptide-resistant enterococci collected from 31 U.S. hospitals in 14 states during May 1988 to July 1992. The isolates included 82 Enterococcus faecium, 8 E. faecalis, 6 Enterococcus spp., 5 E. gallinarum, 3 E. casseliflavus, and 1 E. raffinosus. The isolates were categorized into the following four phenotypes of glycopeptide resistance on the basis of their MIC patterns: (i) 70 VanA (vancomycin [Vm] MIC, > or = 64 micrograms/ml; teicoplanin [Tei] MIC, 16 to > or = 128 micrograms/ml), (ii) 26 VanB (Vm MIC, 16 to 1,024 micrograms/ml; Tei MIC, < or = 2 micrograms/ml), (iii) 5 VanC (Vm MIC, 4 to 16 micrograms/ml; Tei MIC, < or = 2 micrograms/ml) in E. gallinarum, and (iv) 3 E. casseliflavus and 1 E. raffinosus isolates for which Vm MICs were 4 to 16 micrograms/ml and Tei MICs were < or = 1 micrograms/ml were called unclassified. Of the 101 isolates with the VanA, VanB, and VanC phenotypes, 99 were confirmed by production of a specific 1,030-, 433-, or 796-bp polymerase chain reaction product, respectively, and hybridization with the respective gene probe. The vanA gene was also detected in the E. raffinosus isolate for which the Vm MIC was 16 micrograms/ml and the Tei MIC was 1 microgram/ml. The vanA gene was located on either a 34- or a 60-kb plasmid in all of the U.S. isolates examined. Pulsed-field gel electrophoresis demonstrated both intrahospital and interhospital diversity among Vmr enterococci in the United States and was more useful than plasmid analysis for epidemiologic studies.

Streptococcus pneumoniae isolates resistant to penicillin, chloramphenicol, tetracycline and sulfamethoxazole-trimethroprim are being recovered with increasing frequency in the United States. We analyzed the penicillin-binding proteins (PBPs), multilocus enzyme electrophoresis (MLEE) genotypes, and ribotypes of 22 multiresistant serotype 23F isolates of S. pneumoniae from the United States and 1 isolate each from Spain and South Africa. Also included were seven multiresistant isolates of other serotypes, three penicillin-resistant but chloramphenicol-susceptible serotype 23F isolates, and two penicillin-susceptible isolates (one penicillin-susceptible isolate was serotype 23F). Fifteen of the 22 multiresistant isolates from the United States and the isolates from Spain and South Africa had identical PBP patterns, MLEE profiles, and ribotypes. Six of the remaining seven multiresistant isolates were related by PBP pattern, but demonstrated slightly different MLEE and/or ribotype profiles, possibly because of acquisition of additional resistance markers (four of the six isolates were also resistant to erythromycin). The remaining multiresistant serotype 23F isolate had a unique PBP pattern and ribotype and was only distantly related to the other pneumococcal isolates by MLEE analysis. The PBP patterns, MLEE profiles, and ribotypes of the multiresistant serotype 23F isolates were easily distinguished from those of six multiresistant isolates of other serotypes; three other penicillin-resistant, chloramphenicol-susceptible, serotype 23F isolates; and two penicillin-susceptible isolates. One exception was a multiresistant serotype 19A isolate that was highly related to the clonal group by PBP pattern and MLEE analysis and that had a ribotype similar to those of the other erythromycin-resistant serotype 23F isolates. MLEE analysis and ribotyping were more discriminating than were the PBP patterns in discerning strain differences. These data strongly suggest that a multiresistant clone of S. pneumoniae serotype 23F that is related to multiresistant isolates from Spain and South Africa has become disseminated in the United States. Clinicians should be alerted to the spread of these multiresistant strains in the United States.

Since 1988, when the first vancomycin-resistant enterococcus was described, several descriptions of failures of disk diffusion breakpoints to detect low-level vancomycin resistance (MICs, 8 to 32 micrograms/ml) have been published. A four-laboratory collaborative study was undertaken to establish more accurate breakpoints for the disk test. Mueller-Hinton agar was used to perform dilution testing (in three laboratories) and disk diffusion testing (in all laboratories). Results were determined at 18, 24, and 48 h, and zones of inhibition were read using both transmitted and reflected light. One hundred organisms (35 Enterococcus faecalis, 55 E. faecium, and 10 E. gallinarum or E. casseliflavus isolates) were selected to represent vancomycin-susceptible and -resistant phenotypes. Interlaboratory agreement of agar dilution MICs was better at 24 h (91 to 94% within +/- 1 dilution) than at 18 h (76% within +/- 1 dilution). Therefore, 24-h agar dilution MIC results were used as the reference. For disk diffusion, it was critical to note the presence of a haze or colonies inside the zone when interpreting the test, since this correlated better with the results of the agar dilution test. The presence of a haze or inner colonies was best detected by reading the zones with transmitted light and incubating the plates for a full 24 h. When plotted against 24-h agar dilution MICs, breakpoints of /= 17 mm (susceptible) resulted in 58 minor errors (14.5% of total values) and 5 very major errors (2.2% of resistant values or 1.3% of total values). No major errors were seen. Results of repeat testing using a common lot of Mueller-Hinton agar showed 52 minor errors (13.3%) and 4 major errors (4.2% of susceptible values of 1.0% pf total values) but no very major errors. It is recommended that any haze or colonies within the zone be taken into account when determining zones of inhibition and that an MIC test be performed for strains with intermediate zones if vancomycin is being considered for treatment.

We collected 705 isolates of enterococci (1 per patient) from cultures of a variety of anatomic sites from patients at eight tertiary-care hospitals in six geographic regions of the United States. A total of 632 (90%) Enterococcus faecalis, 58 (8%) E. faecium, 5 E. gallinarum, 4 E. avium, 3 E. casseliflavus, 1 E. raffinosus, and 1 E. hirae isolate and 1 biochemical variant of E. faecalis were identified; 606 (86%) of these isolates were associated with clinical infections. The most common sites of isolation were the urinary tract (402 [57%]), nonsurgical wounds (94 [13%]), the bloodstream (74 [10%]), and surgical wounds (62 [9%]). High-level resistance to gentamicin or streptomycin or both was detected in 265 (38%) of the isolates. We identified two E. faecalis isolates resistant to vancomycin (MICs, 32 and 128 micrograms/ml) and 11 beta-lactamase-producing E. faecalis isolates. E. faecium isolates were significantly more resistant than E. faecalis isolates to penicillin, ampicillin, piperacillin, imipenem, and ciprofloxacin (P less than 0.001). The MICs for the 15 non-E. faecalis, non-E. faecium enterococci indicated variable resistance to ciprofloxacin and the penicillins. Antimicrobial susceptibility patterns vary among species of enterococci, and these organisms, while commonly resistant to high-level aminoglycosides, can also acquire resistance to vancomycin or the ability to produce beta-lactamase. Because of these diverse antimicrobial resistance mechanisms, successful treatment and control of enterococcal infections with current antimicrobial agents are becoming increasingly difficult.

A five-laboratory collaborative study was undertaken to determine the precision and accuracy of broth microdilution susceptibility tests of Streptococcus pneumoniae isolates performed with Haemophilus test medium (HTM) compared with tests performed with lysed horse blood-supplemented Mueller-Hinton broth (LHB). The intra-and interlaboratory reproducibilities of MICs of 10 antimicrobial agents determined with the two media were found to be quite similar and highly reproducible in both media. On the basis of favorable performance in this study, S. pneumoniae ATCC 49619 is recommended as a quality control strain to assess the performance of HTM when this medium is used for testing of pneumococci. Testing of 293 unique clinical isolates of S. pneumoniae with both media in the respective participant laboratories allowed a direct comparison of MIC results and a calculation of interpretive error rates. Although there were some slight differences between MICs determined with HTM and MICs determined with LHB, few very major or major errors resulted from testing the clinical isolates against the 10 antimicrobial agents. However, MIC-interpretive criteria specific for S. pneumoniae should be developed and promulgated through a national consensus mechanism.

Objective: To describe the epidemiology of a cluster of vancomycin-resistant Enterococcus faecium (VAREC) in a cardiothoracic surgery intensive care unit., Design: A case series of patients identified through review of surveillance data on nosocomial infections, review of microbiologic records, and culture survey of patients in the unit., Results: Six patients in the cardiothoracic surgery intensive care unit had VAREC with identical antimicrobic susceptibility patterns over a 6-month period. Four patients were identified with VAREC through prospective surveillance and 2 through retrospective review. Prior vancomycin use was seen more commonly in patients with VAREC (6/6, 100%) than in those without VAREC (3/12, 25%) (Fisher's exact test, p = .01). Six of the 7 patients with prior infection developed VAREC (85.7%). A prior nosocomial infection and prior exposure to vancomycin were found to be important variables in a logistic regression analysis. VAREC also was isolated from the environment. A combination of cohorting of patients and staff, and modifications of standard contact isolation practices eliminated the presence of VAREC from the cardiothoracic surgery intensive care unit., Conclusions: The results suggest that prior administration of vancomycin, especially in the patient who develops nosocomial infection, can influence the acquisition of vancomycin-resistant enterococci and that VAREC may be transmitted from patient to patient. Using a modification of the standard infection control practice of isolation, we were able to control the spread of this resistant strain of E faecium.

Eighty-five strains of vancomycin-resistant gram-positive bacteria from three genera, Leuconostoc, Pediococcus, and Lactobacillus, were tested to determine susceptibility to 24 antimicrobial agents by broth microdilution and to 10 agents by disk diffusion. The MICs of vancomycin and teicoplanin ranged from 64 to greater than 512 micrograms/ml; however, the MICs of daptomycin, a new lipopeptide, were all less than or equal to 0.25 micrograms/ml. None of the organisms were resistant to imipenem, minocycline, chloramphenicol, gentamicin, or daptomycin. The MICs of penicillin were in the moderately susceptible range for all but three strains. Susceptibility to the other agents varied by genus and, in some cases, by species. When disk diffusion results were compared with MICs for drugs recommended for streptococci by the National Committee for Clinical Laboratory Standards, Villanova, Pa., few very major or major errors were obtained, but the number of minor errors was 19.3%. Therefore, we recommended that MIC testing be used instead of disk diffusion testing for these organisms.

Aerococci can be misidentified as streptococci, enterococci, pediococci, lactococci, or leuconostocs. To distinguish the genus and determine if another species is needed in the present taxon, we analyzed 37 aerococci for cellular fatty acids and compared them with 377 strains of gram-positive cocci, including the species type strains from each of the related genera. The cellular fatty acid profile of aerococci was distinguishable from other genera. Two relatively novel fatty acids found in the aerococci were identified as C16:1 omega 9c and C16:1 omega 9t. Eleven strains of aerococci (including a strain originally identified as "Gaffkya" species) were chosen for DNA-DNA reassociation studies with the type strain Aerococcus viridans ATCC 11563; DNAs from eight of these strains were more than 75% related to the type strain and had 1 to 4% divergence in related sequences. The remaining three strains were 60 to 70% related to the type strain, had 7 to 11.5% divergence, and may represent a second species, Aerococcus genospecies 2. beta-Glucuronidase, alpha-galactosidase, and beta-galactosidase were useful in characterizing the aerococci.