Characterization of Staphylococci with Reduced Susceptibilities to Vancomycin and Other Glycopeptides

During the last several years a series of staphylococcal isolates that demonstrated reduced susceptibility to vancomycin or other glycopeptides have been reported. We selected 12 isolates of staphylococci for which the vancomycin MICs were ≥4 μg/ml or for which the teicoplanin MICs were ≥8 μg/ml and 24 control strains for which the vancomycin MICs were ≤2 μg/ml or for which the teicoplanin MICs were ≤4 μg/ml to determine the ability of commercial susceptibility testing procedures and vancomycin agar screening methods to detect isolates with reduced glycopeptide susceptibility. By PCR analysis, none of the isolates with decreased glycopeptide susceptibility contained known vancomycin resistance genes. Broth microdilution tests held a full 24 h were best at detecting strains with reduced glycopeptide susceptibility. Disk diffusion did not differentiate the strains inhibited by 8 μg of vancomycin per ml from more susceptible isolates. Most of the isolates with reduced glycopeptide susceptibility were recognized by MicroScan conventional panels and Etest vancomycin strips. Sensititre panels read visually were more variable, although with some of the panels MICs of 8 μg/ml were noted for these isolates. Vitek results were 4 μg/ml for all strains for which the vancomycin MICs were ≥4 μg/ml. Vancomycin MICs on Rapid MicroScan panels were not predictive, giving MICs of either ≤2 or ≥16 μg/ml for these isolates. Commercial brain heart infusion vancomycin agar screening plates containing 6 μg of vancomycin per ml consistently differentiated those strains inhibited by 8 μg/ml from more susceptible strains. Vancomycin-containing media prepared in-house showed occasional growth of susceptible strains, Staphylococcus aureus ATCC 29213, and on occasion, Enterococcus faecalis ATCC 29212. Thus, strains of staphylococci with reduced susceptibility to glycopeptides, such as vancomycin, are best detected in the laboratory by nonautomated quantitative tests incubated for a full 24 h. Furthermore, it appears that commercial vancomycin agar screening plates can be used to detect these isolates.     

Evaluation of discrepancies between oxacillin and cefoxitin disk susceptibility for detecting methicillin-resistant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The Clinical Laboratory Standards Institute recommends that if both cefoxitin and oxacillin are tested against Staphylococcus aureus and either result is measured as resistant, the organism should be reported as oxacillin resistant. This indicates that discrepancies may be present between oxacillin and cefoxitin sensitivities in S. aureus. In this study, we aimed to investigate the discrepancy between oxacillin and cefoxitin susceptibility in S. aureus clinical isolates. Of 10,980 S. aureus isolates recovered from 2005 to 2010, 27 (0.3%) isolates with discordant results between oxacillin and cefoxitin were collected. Fourteen (oxacillin diameters 10–12 mm) of the 27 strains were susceptible (MICs = 0.5–2 μg/ml) and 13 (6–13 mm) were resistant (4–>256 μg/ml) to oxacillin. The cefoxitin MICs of 14 oxacillin-susceptible and 13 oxacillin-resistant strains ranged between 4 and 8 and 8 to 32 μg/ml, respectively. Discrepancies were present between oxacillin and cefoxitin in S. aureus, and these strains should be further tested for oxacillin MICs and for the mecA gene or β-lactamase activity.     

In vitro selection of resistance to vancomycin in bloodstream isolates ofStaphylococcus haemolyticus andStaphylococcus epidermidis

The purpose of the study was to determine whether vancomycin-resistant strains ofStaphylococcus haemolyticus could be selected regardless of the initial MIC of vancomycin. Twenty-one bloodstream isolates ofStaphylococcus haemolyticus were studied by broth and agar selection methods. The broth method selected strains for which MICs of vancomycin ranged from 4 to 32 µg/ml and MBCs from 16 to>128 µg/ml. The agar method selected strains for which MICs ranged from 8 to 32 µg/ml and MBCs from 8 to>128 µg/ml. For comparison, seven strains ofStaphylococcus epidermidis were evaluated by the agar selection method. Final MICs of vancomycin ranged from 8 to 16 µg/ml; MBCs ranged from 16 to 64 µg/ml. Clearly, in vitro exposure to vancomycin can select strains ofStaphylococcus haemolyticus andStaphylococcus epidermidis for which MIC values are beyond the susceptible breakpoint.     

Comparative assessment of inoculum effects on the antimicrobial activity of amoxycillin-clavulanate and piperacillin-tazobactam with extended-spectrum β-lactamase-producing and extended-spectrum β-lactamase-non-producing Escherichia coli isolates.

A significant inoculum-size effect has been observed with piperacillin-tazobactam, and has been associated with β-lactamase production in extended-spectrum β-lactamase (ESBL) producers. This association has not been previously studied in the case of amoxycillin-clavulanate. Piperacillin-tazobactam and amoxycillin-clavulanate were compared, using high inocula of susceptible strains either harbouring ESBLs or not. Two non-ESBL-producing and 15 amoxycillin-clavulanate-susceptible and piperacillin-tazobactam-susceptible ESBL-producing Escherichia coli isolates, and their respective transconjugants, were tested in dilution susceptibility tests using standard and 100-fold higher inocula. Three ESBL-producing strains and E. coli ATCC 25922 were selected for time-kill studies using standard and high initial inocula. At high inocula, MICs of piperacillin increased >eight-fold for non-ESBL-producing strains, and MICs of piperacillin-tazobactam (8 : 1 ratio or with tazobactam fixed at 4 mg/L) increased>eight-fold for all ESBL-producing strains. However, amoxycillin MICs were not affected by a high inoculum with non-ESBL-producing strains, whereas the MICs of amoxycillin-clavulanate (2 : 1 and 4 : 1) increased ≤four-fold for ESBL producers, using the broth and agar dilution methods. In kinetic studies at a high inoculum, amoxycillin and amoxycillin-clavulanate were bactericidal against E. coli ATCC 25922, whereas piperacillin and piperacillin-tazobactam yielded decreases of <1 log₁₀ CFU/mL. Similarly, at a high inoculum, only amoxycillin-clavulanate was able to maintain bactericidal rates of killing over 24 h against the ESBL-positive E. coli isolates. The stability of amoxycillin-clavulanate and the contrasting results obtained with piperacillin-tazobactam against high inocula of ESBL-non-producing and ESBL-producing E. coli strains appear to be related to aspects other than the amount of β-lactamase production.     

In vitro activity of ciprofloxacin against methicillin-susceptible and methicillin-resistant staphylococci

Ciprofloxacin was found to be active against selected strains of methicillin-resistant as well as methicillin-susceptible Staphylococcus aureus and Staphylococcus epidermidis, having minimal inhibitory concentrations <0.8 g/ml with inocula of approximately 10⁴ CFU/ml. When killing kinetic studies were done with inocula of approximately 10⁶ CFU/ml and various ciprofloxacin concentrations, the killing effect was low (<3 log₁₀ in 6h) and regrowth of the culture was seen after 24 h in at least one Staphylococcus aureus strain. After 48 h incubation, mutants with MICs eightfold that of the parental strain had grown to a number of approximately 10¹⁰ CFU/ml.     

Synergy of β-Lactams with Vancomycin against Methicillin-Resistant Staphylococcus aureus: Correlation of Disk Diffusion and Checkerboard Methods

Modified disk diffusion (MDD) and checkerboard tests were employed to assess the synergy of combinations of vancomycin and β-lactam antibiotics for 59 clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA) and Mu50 (ATCC 700699). Bacterial inocula equivalent to 0.5 and 2.0 McFarland standard were inoculated on agar plates containing 0, 0.5, 1, and 2 μg/ml of vancomycin. Oxacillin-, cefazolin-, and cefoxitin-impregnated disks were applied to the surface, and the zones of inhibition were measured at 24 h. The CLSI-recommended checkerboard method was used as a reference to detect synergy. The MICs for vancomycin were determined using the Etest method, broth microdilution, and the Vitek 2 automated system. Synergy was observed with the checkerboard method in 51% to 60% of the isolates when vancomycin was combined with any β-lactam. The fractional inhibitory concentration indices were significantly lower in MRSA isolates with higher vancomycin MIC combinations (P < 0.05). The overall agreement between the MDD and checkerboard methods to detect synergy in MRSA isolates with bacterial inocula equivalent to McFarland standard 0.5 were 33.0% and 62.5% for oxacillin, 45.1% and 52.4% for cefazolin, and 43.1% and 52.4% for cefoxitin when combined with 0.5 and 2 μg/ml of vancomycin, respectively. Based on our study, the simple MDD method is not recommended as a replacement for the checkerboard method to detect synergy. However, it may serve as an initial screening method for the detection of potential synergy when it is not feasible to perform other labor-intensive synergy tests.     

In vitro activity of FCE 22101, imipenem,and ceftazidime against over 6,000 bacterial isolates and MIC quality control limits of FCE 22101

Six geographically separate laboratories within the USA tested 6,198 bacterial isolates against FCE 22101 (a penem), imipenem (a carbapenem) and ceftazidime (a third-generation cephalosporin). Ninety-three percent of 2,749Enterobacteriaceae were inhibited by FCE 22101, while 95 % were susceptible to ceftazidime and 99 % were susceptible to imipenem. FCE 22101 had little activity againstPseudomonas spp. but was active against most gram-positive pathogens, including enterococci. FCE 22101 MICs for standard quality control strains were defined as 0.5–2.0µg/ml forEscherichia coli ATCC 25922, 2–8µg/ml forEnterococcus faecalis ATCC 29212 and 0.06–0.25µg/ml forStaphylococcus aureus ATCC 29213.     

Comparative Activity of Eighteen Antimicrobial Agents against Anaerobic Bacteria Isolated in South Africa

 The in vitro activity of 18 antimicrobial agents was determined against 378 anaerobic bacteria isolated in Bloemfontein, South Africa, during 1996/97. Against the gram-positive isolates, MICs of penicillin and cefoxitin were >0.5 μg/ml and >16 μg/ml, respectively, for five and three strains of non-perfringens Clostridium spp. Seventeen Peptostreptococcus anaerobius strains were resistant to penicillin (MIC≥2 μg/ml). All gram-positive anaerobes tested except one Peptostreptococcus sp. and one Clostridium sp. were susceptible to dalfopristin-quinupristin (MICs≤1 μg/ml). The carbapenems exhibited excellent activity against the gram-positive isolates and were effective against most gram-negative anaerobes, with the exception of the fusobacteria. Only seven strains exhibited decreased susceptibility to trovafloxacin (MICs>2 μg/ml). In mixed anaerobic/aerobic infections, carbapenems and the fourth-generation quinolone trovafloxacin were the agents most suitable for us as broad-spectrum monotherapy.     

Effect of pathological changes of pH,pO2 and pCO2 on the activity of antimicrobial agents in vitro

Since standard susceptibility tests reflect the physiological rather than the pathological conditions prevailing within an infected abdomen, as recently documented, the effect of reduced pH and pO₂ and increased pCO₂ on the activity of antibiotics in vitro was studied. MICs were determined in vitro under standard culture conditions (MIC_standard) and modified conditions (MIC_modification) simulating the previously determined pathological values. Various classes of antibiotics were affected differently by the modified conditions. However, within an antibiotic class similar results were obtained for gram-negative and gram-positive pathogens. Median MIC_modification/MIC_standard ratios were 4 for aminoglycosides, 2 for quinolones and clindamycin, 1 for cephalosporins, and 0.5 for penicillins and vancomycin. Anaerobic conditions and a pH of 6.4 further increased the ratio of aminoglycosides to 8. Ratios were similar within an antibiotic class at inocula of 10⁵ or 10⁷ cfu/ml. All MICs determined in tests with imipenem against gram-negative and gram-positive bacteria and with vancomycin against gram-positive organisms were below the susceptibility breakpoint, whatever conditions and inocula were employed. In contrast, the percentage of MICs in susceptibility range using high inocula and modified conditions decreased to 78 % for penicillins, 73 % for cephalosporins, 22 % for aminoglycosides, 11 % for quinolones and 0 % for clindamycin. In conclusion, routine susceptibility testing may overestimate the activity of aminoglycosides and underestimate the activity of beta-lactams under the conditions prevailing during abdominal infection.     

Quality control limits for teicoplanin susceptibility tests and confirmation of disk diffusion interpretive criteria.

For monitoring the performance of teicoplanin susceptibility tests, the following quality control limits are recommended: Staphylococcus aureus ATCC 29213, MIC of 0.12 to 0.5 micrograms/ml; Enterococcus faecalis ATCC 29212, MIC of 0.06 to 0.25 micrograms/ml; and S. aureus ATCC 25923, zones 15 to 19 mm in diameter (30-micrograms disks). However, some lots of Mueller-Hinton agar provided unusually large zones of inhibition with both vancomycin and teicoplanin disks, and these lots should be excluded before routine use. Teicoplanin and vancomycin differed only in their activity against oxacillin-resistant strains of Staphylococcus haemolyticus, which had decreased susceptibility to teicoplanin but were fully susceptible to vancomycin. For tests with 30-micrograms teicoplanin disks, zones less than or equal to 10 and greater than or equal to 14 mm in diameter represent resistant and susceptible breakpoints, respectively.     

Antibacterial effects of silver ions on growth of gram-negative bacteria and biofilm formation

The average minimal inhibiting concentrations (MICs) of AgNO₃ for the gram-negative bacteria Escherichia coli K12, Serratia proteamaculans 94, and Serratia liquefaciens MG1 were found to be 0.075–0.3 μg/ml and, for Pseudomonas aeruginosa PAO1 and P. chlororaphis 449, this concentration was 0.15–0.3 μg/ml. The formation of a biofilm by Escherichia coli AB1157 and S. proteamaculans 94 was completely inhibited by 0.3 μg/ml of AgNO₃ and, in the case of Pseudomonas aeruginosa PAO1, biofilm formation was inhibited by 0.6 μg/ml of AgNO₃. Mutations in E. coli genes that encode global regulators of gene expression, such as sigma S and sigma N subunits of RNA polymerase, the catabolite repression protein CRP, and the Lon protease, had no marked effect on the sensitivity of cells to silver. Wild-type E. coli strains and strains deficient in excision repair (uvrA and uvrB), SOS repair, and recombination (recA, lexA, recBC, and recF mutants) did not differ in their sensitivity to silver. This suggests that the sensitivity of the bacteria to the silver was not associated with DNA lesions that could be repaired by these repair and recombination systems. E. coli mutant strains deficient in OmpF or OmpC porins were three to four times more resistant to silver ions than the wild-type strain. Experiments with the pME6863 plasmid, which bears the gene of N-acyl-homoserine lactonase AiiA, demonstrated that the quorum sensing (QS) regulation did not participate in controlling the sensitivity of S. proteamaculans 94 and P. chlororaphis 449 to silver. The same conclusion was drawn from a comparison of AgNO₃ MICs of the S. liquefaciens wild-type strain and a mutant strain deficient in QS.     

Use of the Polymerase Chain Reaction for Rapid Detection of High-Level Mupirocin Resistance in Staphylococci

 The minimum inhibitory concentrations (MICs) of mupirocin were determined by the E test (AB Biodisk, Sweden) and the agar dilution method for 107 staphylococci. The organisms consisted of 34 coagulase-negative staphylococci and 73 methicillin-resistant Staphylococcus aureus. Polymerase chain reaction (PCR) primers designed to amplify a 456 bp region of the plasmid-borne isoleucyl tRNA synthetase gene (ileS–2), responsible for high-level mupirocin resistance in staphylococci, were used on DNA preparations from these isolates. Isolates with high-level mupirocin resistance due to the ileS–2 gene should be PCR positive. There was close correlation between the E test and agar dilution MIC values, with only two strains differing by more than two serial dilutions. Most (51 of 54 strains) of the high-level resistant strains (MIC>256 μg/ml) were resistant to the highest concentration of mupirocin tested (1024 μg/ml). PCR correctly classified all but four (96%) of the isolates in accordance with the results of agar dilution. All four isolates that gave discrepant results were methicillin-resistant Staphylococcus aureus. Two of these were PCR positive, yet the MIC of mupirocin for these strains was <0.06 μg/ml; on prolonged incubation they produced halos within the inhibition zone on agar diffusion testing, suggesting that the phenotypic results may have been erroneous. One of 54 isolates for which the MIC exceeded 256 μg/ml was PCR negative when tested by the original methodology, but a 456 bp product was produced when retested using a lowered annealing temperature. One isolate for which the MIC of mupirocin was 16 μg/ml by agar dilution and 8 μg/ml by the E test was positive by PCR. PCR of the ileS–2 gene is a useful, rapid method for detecting high-level mupirocin resistance in staphylococci.     

Postantibiotic and bactericidal effect of imipenem againstPseudomonas aeruginosa

The postantibiotic effect of imipenem onPseudomonas aeruginosa was studied at different inocula using one ATCC strain and four clinical isolates. The postantibiotic effect was measured using two different methods: viable counts and bioluminescence assay of intracellular bacterial ATP. The postantibiotic effect could be demonstrated with both methods (viable counts 1–2 h, ATP assay 3–5 h) for all strains at an inoculum of 10⁶ CFU/ml. When the inoculum was raised to 10⁸ CFU/ml, no postantibiotic effect could be observed with either method using routine growth conditions. This disappearance of the postantibiotic effect coincided with a loss of bactericidal effect of imipenem when high inocula were used. Improved oxygenation of the cultures restored the bactericidal and postantibiotic effects of imipenem at high inocula.     

Resistance Pattern of Streptococcus pneumoniae in Children During a Four-Year Period in Greece

 The resistance pattern of 432 Streptococcus pneumoniae strains isolated from children with various infections over a 4-year period (1992–1995) was determined. The rates of resistance to penicillin, chloramphenicol, tetracycline, trimethoprim-sulfamethoxazole, erythromycin, clindamycin, cefotaxime, and vancomycin were 10%, 2.8%, 4.6%, 4.9%, 4.4%, 2.5%, 0.9%, and 0%, respectively. All strains not susceptible to penicillin were intermediately susceptible to penicillin-(MIC >0.06–≤1 μg/ml). Isolates not susceptible to penicillin were encountered significantly more often in children with localized infections than in those with invasive disease; these isolates displayed significantly lower susceptibility to non-β-lactam agents as compared with their penicillin-susceptible counterparts.     

In Vitro Evaluation of the Antibacterial Activity of Three Different Central Venous Catheters Against Gram-Positive Bacteria

The aim of this study was to evaluate the activity of three different catheters against Staphylococcus aureus ATCC 29213 and the slime-producing Staphylococcus epidermidis ATCC 35984 (RP62A). Three central venous catheters were evaluated: one impregnated with silver sulfadiazine–chlorhexidine, one to which minocycline/rifampin is bonded and a novel one into which silver, platinum and carbon are incorporated. A nonantiseptic catheter was used as the control catheter. One-centimeter trisected pieces of catheter were immersed in phosphate-buffered saline (0.01 mol/l) with 0.25% dextrose and incubated. On days 1, 3, 7, 14 and 21, a 1 ml standardized inoculum was added for 30 min and then replaced with phosphate-buffered saline with 0.25% dextrose. One-third of the samples were immediately sonicated and plated to determine bacterial adherence. The remaining segments were incubated for 4 and 24 h to determine the persistence of bacterial adherence. Bacterial adherence to the catheters impregnated with silver sulfadiazine-chlorhexidine was reduced 91–98% for the first 7 days. Adherence of Staphylococcus aureus to catheters into which silver, platinum and carbon are incorporated was reduced 70% on day 1 and 35% on day 3. Adherence to minocycline/rifampin-bonded catheters was quite variable. There was an 85.6–99.8% reduction in the persistence of bacterial adherence to the three catheters compared to controls. Bacteriostatic and bactericidal studies indicated that the effluents from the catheters impregnated with silver sulfadiazine-chlorhexidine were bactericidal, while effluents from the minocycline/rifampin-bonded catheters were bacteriostatic. The antibacterial activity of the effluents from catheters impregnated with silver sulfadiazine-chlorhexidine dissipated by day 7, while the activity of effluents from the minocycline/rifampin-bonded catheters continued to show activity at day 21. No measurable antibacterial activity was detected in the effluents of the catheters into which silver, platinum and carbon are incorporated. These data suggest that catheters coated with antibiotic/antibacterial agents and the novel catheters that incorporate antiseptic agents have different activities against initial bacterial adherence. All of them, however, effectively prevent bacterial colonization by gram-positive bacteria. Electronic Publication     

Effects of Aggregate and Individual Antibiotic Exposure on Vancomycin MICs for Staphylococcus aureus Isolates Recovered from Pediatric Patients

We evaluated the evolution of vancomycin MICs for Staphylococcus aureus and their relationship with vancomycin use among hospitalized children. S. aureus isolates recovered from sterile sites were prospectively tested for vancomycin susceptibility using the Etest between 1 April 2000 and 31 March 2008. Vancomycin MICs were grouped into three categories: ≤1, 1.5, and 2 μg/ml. The association between vancomycin MICs and aggregate vancomycin use and individual patient vancomycin exposure 6 months prior to the documented infection was assessed. The geometric mean values for vancomycin MICs for S. aureus fluctuated over time without a significant trend (P = 0.146). Of the 436 patients included in the study, 363 (83%) had methicillin-susceptible S. aureus (MSSA) and 73 (17%) had methicillin-resistant S. aureus (MRSA) infections. The rate of isolates with a vancomycin MIC of 2 μg/ml increased from 4% (2 of 46) in 2000 to 2001 to 24% (11 of 46) in 2007 to 2008, despite a decrease in vancomycin use (r = −0.11; P = 0.825). The percentage of isolates with a vancomycin MIC of 2 μg/ml was higher for MRSA (15%; 11 of 73) than for MSSA strains (5.2%; 19 of 363) (χ² = 9.2; P = 0.01). Individual patient vancomycin exposure was not associated with a higher vancomycin MIC. In the unadjusted model, in which we compared patients with S. aureus infections with MICs of ≤1 μg/ml, the odds ratios of exposure rates for patients with isolates with MICs of 1.5 μg/ml and 2 μg/ml were 1.02 (P = 0.929) and 1.13 (P = 0.767), respectively. In our experience, the geometric means of vancomycin MICs from S. aureus isolates recovered from hospitalized children oscillated over time and were not associated with previous individual patient vancomycin exposure or aggregate vancomycin use.     

Accuracy of Commercial and Reference Susceptibility Testing Methods for Detecting Vancomycin-Intermediate Staphylococcus aureus

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, ≤1 μg/ml [n = 60 strains], 2 μg/ml [n = 24], 4 μg/ml [n = 36], or 8 μg/ml [n = 9]) were selected from the Centers for Disease Control and Prevention strain collection. The results of BMD with Difco Mueller-Hinton broth were used as the standard for data analysis. Essential agreement (percent ±1 dilution) ranged from 98 to 100% for all methods except the method with the Vitek Legacy system, for which it was 90.6%. Of the six commercial MIC systems tested, the Sensititre, Vitek Legacy, and Vitek 2 systems tended to categorize VISA strains as susceptible (i.e., they undercalled resistance); the MicroScan and Phoenix systems and Etest tended to categorize susceptible strains as VISA; and the Vitek Legacy system tended to categorize VISA strains as resistant (i.e., it overcalled resistance). Disk diffusion categorized all VISA strains as susceptible. No susceptible strains (MICs ≤ 2 μg/ml) grew on the VScr, but all strains for which the VA MICs were 8 μg/ml grew on the VScr. Only 12 (33.3%) strains for which the VA MICs were 4 μg/ml grew on VScr. The differentiation of isolates for which the VA MICs were 2 or 4 μg/ml was difficult for most systems and methods, including the reference methods.In January 2006, the Clinical and Laboratory Standards Institute (CLSI) published new interpretive criteria for vancomycin and Staphylococcus aureus. The breakpoints were lowered from ≤4 μg/ml to ≤2 μg/ml for susceptible, 8 to 16 μg/ml to 4 to 8 μg/ml for intermediate, and ≥32 μg/ml to ≥16 μg/ml for resistant (2). The vancomycin breakpoints for coagulase-negative staphylococci were not changed. The rationale for lowering the S. aureus intermediate breakpoint to 4 μg/ml was (i) that intermediate S. aureus isolates, although they are rare, likely represented a population of organisms that demonstrate heteroresistance, and (ii) limited outcome data suggested that infections with these isolates are likely to fail vancomycin therapy (9). The results of broth microdilution performed by use of the CLSI reference method were the primary S. aureus susceptibility data evaluated before the CLSI breakpoint change was made. We undertook the study described here to determine the accuracy of commercial systems and reference methods for the detection of decreased vancomycin susceptibility among isolates of S. aureus.(This work was presented in part at the 47th Interscience Conference on Antimicrobial Agents and Chemotherapy, Chicago, IL, 17 to 20 September 2007.)     

Antimicrobial activity of heparin

Single clinical isolates of eight species of microorganisms were incubated in solutions of heparin and brain heart infusion broth at various concentrations to determine the possible antibacterial effect of heparin. At heparin concentrations ranging from 12.5 to 400 U/ml, the effect on Escherichia coli, Pseudomonas aeruginosa, Candida albicans, Staphylococcus aureus, and S. epidermidis varied with brain heart infusion broth concentrations of 1.2 to 10% and inocula of 10² to 10⁶ colony-forming units per ml; similar effects were not observed with Klebsiella pneumoniae, Enterobacter aerogenes, and Citrobacter spp. The minimal inhibitory concentrations of heparin for ten strains of each species were determined in 2.5% brain heart infusion broth with inocula of 10⁴ colony-forming units per ml. All 10 isolates of S. aureus and all 10 of S. epidermidis were inhibited by heparin concentrations of 125 to 500 U/ml. Three E. coli, four P. aeruginosa, and nine C. albicans strains were inhibited by ≤500 U of heparin per ml. None of the K. pneumoniae, E. aerogenes, Enterobacter cloacae, and Citrobacter spp. was inhibited by heparin at ≤500 U/ml. Heparin inhibition of S. aureus in 2.5% brain heart infusion broth-500 U of heparin per ml could be quantitatively overcome by addition of magnesium, calcium, or magnesium and calcium. These data suggest that the growth of microorganisms from heparin-containing material may be suppressed.     

Determinants for the Development of Oropharyngeal Colonization or Infection by Fluconazole-Resistant Candida Strains in HIV-Infected Patients

 A point prevalence study to document oral yeast carriage was undertaken. Risk factors for the development of oropharyngeal colonization or infection by fluconazole-resistant Candida strains in HIV-infected patients were investigated with a case-control design. Cases included all patients with fluconazole-resistant strains (MIC≥64 μg/ml), and controls were those with susceptible (MIC≤8 μg/ml) or susceptible-dependent-upon-dose (MIC 16–32 μg/ml) strains. One hundred sixty-eight Candida strains were isolated from 153 (88%) patients, 28 (16%) of whom had oropharyngeal candidiasis. Overall, 19 (12%) of the patients harbored at least one resistant organism (MIC≥64 μg/ml). Among patients with resistant strains, tuberculosis (P<0.001), esophageal candidiasis (P=0.001), clinical thrush (P<0.001), and a CD4+ cell count <200/mm³ (P=0.03) were more frequent. These patients had also been treated more commonly with antituberculous drugs (adjusted odds ratio [OR] 6.13; 95% confidence interval [CI] 2.11–17.80), ciprofloxacin (OR 6.0; 95% CI 1.23–29.26), fluconazole (OR 4.59; 95% CI 1.55–13.52), and steroids (OR 4.13; 95% CI 1.11–15.39). Multivariate analysis showed that the determinants for fluconazole resistance were therapy with antituberculous drugs (OR 3.61; 95% CI 1.08–12.07;P=0.03) and one of the following: previous tuberculosis (OR 3.53; 95% CI 1.08–14.57;P=0.03) or fluconazole exposure (OR 3.41; 95% CI 1.10–10.54). Findings from this study indicate that treatment with antituberculous drugs, previous tuberculosis, and fluconazole exposure are the strongest determinants for development of oropharyngeal colonization or infection by fluconazole-resistant Candida strains in HIV-infected patients.     

Meropenem Alone and in Combination with Vancomycin in Experimental Meningitis Caused by a Penicillin-Resistant Pneumococcal Strain

 In a rabbit model of meningitis caused by a pneumococcus highly resistant to penicillin (MIC, 4 μg/ml), meropenem, a broad-spectrum carbapenem, was bactericidal (–0.48±0.14 Δlog₁₀ cfu/ml·h) and slightly superior to ceftriaxone (–0.34±0.23 Δlog₁₀ cfu/ml·h) and vancomycin (–0.39±0.19 Δlog₁₀ cfu/ml·h). Although the combination of vancomycin with ceftriaxone was significantly more active than ceftriaxone alone (–0.55±0.19 Δlog₁₀ cfu/ml·h), only an insignificant gain was observed by the addition of vancomycin to meropenem (–0.55±0.28 Δlog₁₀ cfu/ml·h).