High-Level Mupirocin Resistance among Spanish Methicillin-Resistant Staphylococcus aureus

 Twelve strains of methicillin-resistant Staphylococcus aureus with high-level resistance to mupirocin were studied. The MIC of methicillin was 16 to 128 mg/l and the MIC of mupirocin ≥512 mg/l. All of the isolates carried a plasmid of about 30 MDa, and one strain lost resistance to mupirocin when the plasmid was cured. Three different resistance patterns were found: six strains were resistant to ciprofloxacin, kanamycin, and 10 mg/l of cadmium sulfate; two strains were resistant to these agents as well as to gentamicin, erythromycin, clindamycin, mercury chloride, and ethidium bromide; and four strains were resistant to the previously named agents as well as to rifampicin and tetracycline. The use of this valuable topical antibiotic, especially in long-term-care facilities, should be monitored to avoid dissemination of resistance.     

New Colorimetric Microdilution Method for In Vitro Susceptibility Testing of Borrelia burgdorferi Against Antimicrobial Substances

 A newly developed colorimetric microdilution method was used to analyze the activity of 12 antimicrobial agents against nine Borrelia burgdorferi isolates, including all three genospecies pathogenic for humans. In addition, in vitro antimicrobial resistance patterns of Borrelia valaisiana and Borrelia bissettii tick isolates were investigated. The applied test system is based upon color changes that occur in the presence of phenol red and result from the accumulation of nonvolatile acid produced by actively metabolizing spirochetes. After 72 h of incubation, minimal inhibitory concentrations (MICs) were determined from the decrease of absorbance by software-assisted calculation of growth curves. MIC values were lowest for azlocillin (MIC, ≤0.125 μg/ml), ceftriaxone (MIC range, ≤0.015–0.06 μg/ml), and azithromycin (MIC range, ≤0.015–0.06 μg/ml). Whereas tobramycin (MIC range, 8–64 μg/ml) exhibited little activity, spectinomycin (MIC range, 0.25–2 μg/ml) showed in vitro antimicrobial activity against Borrelia burgdorferi. The MICs of penicillin G for Borrelia afzelii isolates were ten times higher than those for Borrelia burgdorferi, Borrelia valaisiana, and Borrelia bissettii isolates (P<0.05) and 100 times higher than those for isolates belonging to the genospecies Borrelia garinii (P<0.05). Further significant differences with respect to the MIC values of the other antimicrobial agents tested were not noted. The colorimetric microdilution method offered the advantages of reliability, reproducibility, and convenience and could handle large numbers of isolates and antibiotics.     

In Vitro Susceptibility to Gemifloxacin and Trovafloxacin of Streptococcus pneumoniae Strains Exhibiting Decreased Susceptibility to Ciprofloxacin

 The in vitro susceptibility to trovafloxacin and gemifloxacin of Streptococcus pneumoniae strains exhibiting decreased susceptibility to ciprofloxacin (MIC ≥2 μg/ml; 30 strains with intermediate resistance [MIC 2 μg/ml] and 43 strains with complete resistance [MIC ≥4 μg/ml]) was determined. Seventy-three strains collected in a surveillance study carried out from May 1996 to April 1997 in Spain (prior to commercialisation of trovafloxacin and gemifloxacin) from patients with respiratory tract infections were tested. The antibacterial activity of gemifloxacin was affected to a lesser extent than that of trovafloxacin by the increase in the MIC of ciprofloxacin, with gemifloxacin showing significantly (P≤0.001) better antibacterial activity than trovafloxacin in all ciprofloxacin MIC categories (MIC50/MIC90 values of 0.015/0.03, 0.015/0.06, 0.03/0.06 and 0.12/0.25 μg/ml for gemifloxacin vs 0.12/0.12, 0.12/1, 0.25/0.5 and 2/4 μg/ml for trovafloxacin in the 2, 4, 8 and ≥16 μg/ml ciprofloxacin MIC categories, respectively). Nine (12.3%) of these 73 strains exhibited decreased susceptibility to trovafloxacin (≥2 μg/ml), whereas all strains were inhibited by 0.25 μg/ml of gemifloxacin.     

Comparative In Vitro Activity of Quinolones Against Stenotrophomonas maltophilia

 The susceptibility of 109 Stenotrophomonas maltophilia isolates, all characterized by pulsed-field gel electrophoresis, to nine quinolones was studied. Grepafloxacin, trovafloxacin, and moxifloxacin displayed similar intrinsic activities (MIC90, 0.5 μg/ml), which were lower than those of ofloxacin and ciprofloxacin (MIC90, 4 μg/ml), norfloxacin (MIC90, 64 μg/ml), and nalidixic acid (MIC90, 32 μg/ml). Nalidixic acid was generally one- to twofold dilutions more active than norfloxacin. According to the criteria of the National Committee for Clinical Laboratory Standards (NCCLS), the percentage of isolates susceptible to ciprofloxacin (breakpoint ≤1 μg/ml) was 76.1%. Using the NCCLS breakpoint for comparative purposes, the percentage of isolates susceptible to grepafloxacin, moxifloxacin, and trovafloxacin was 95.4, 96.4, and 96.4%, respectively. These results indicate that new quinolones may potentially be used for the management of Stenotrophomonas maltophilia infections.     

Characterization of Isoniazid-Resistant Strains of Mycobacterium tuberculosis on the Basis of Phenotypic Properties and Mutations in katG

 Forty isoniazid-resistant Mycobacterium tuberculosis isolates were characterized on the basis of phenotypic properties (i.e., catalase activity, MIC of isoniazid, and growth pattern in the presence of 7 different concentrations of isoniazid) and alterations in the katG gene (codons 315 and 463). Three different growth patterns could be distinguished: concentration-dependent inhibition of growth was observed in 29 strains, similar growth at all concentrations was seen in 7 strains, and enhanced growth at low concentrations of isoniazid was evident in 4 strains. The MIC of isoniazid was ≤4 μg/ml for 29 of 40 strains. Mutation at codon 315 of the katG was detected in 28 of 40 strains. However, only one of the seven strains for which the MIC of isoniazid was ≥16 μg/ml had mutation at this codon. Five of these seven strains for which the MIC was ≥16 μg/ml had no catalase activity. The results indicate that the MIC of isoniazid for a majority of strains is below the level achievable in serum. Therefore, isoniazid may be beneficial for the treatment of some cases of multidrug-resistant tuberculosis. Determination of catalase activity aids in the detection of isolates for which MICs are high and could, in conjunction with molecular methods, provide rapid detection of most isoniazid-resistant strains.     

Urinary Excretion and Bactericidal Activity of Intravenous Ciprofloxacin Compared with Oral Ciprofloxacin

 Twelve healthy volunteers participated in a randomized crossover study to compare urinary concentrations, serum parameters, and urinary bactericidal activity of ciprofloxacin after single intravenous (i.v.) doses of 200 mg and 400 mg and an oral (p.o.) dose of 500 mg. The median serum concentrations at 1 h after administration were 1 μg/ml, 4.3 μg/ml, and 2.2 μg/ml, respectively. Between the first collection period (0–2 h) and the last collection period (38–48 h), the median urinary concentrations decreased from 394 μg/ml, 675 μg/ml, and 585 μg/ml, respectively, to 0.3 μg/ml, 0.6 μg/ml, and 1 μg/ml, respectively. The urinary concentrations after the 400 mg i.v. and the 500 mg p.o. doses were not statistically different but were significantly higher than those after the 200 mg i.v. dose. The urinary bactericidal titers (UBTs), defined as the highest urinary dilution bactericidal for the organism tested, were determined against Escherichia coli (ATCC 25922) and eight uropathogens up to 48 h after administration of ciprofloxacin. The UBTs after the 400 mg i.v. and the 500 mg p.o. doses were similar and were significantly higher (P<0.05) than those following the 200 mg i.v. dose. After 400 mg i.v. and 500 mg p.o., median UBTs of ≥1 : 4 were present up to 48 h for all strains for which the MIC was ≤0.5 μg/ml, except for one nalidixic-acid resistant Escherichia coli strain for which the MIC was 0.25 μg/ml. Species for which the MIC is ≥1 μg/ml showed median UBTs of ≥1 : 4 for 8–16 h. Median UBTs of ≥1 : 4 were present up to 8 and 12 h for both Pseudomonas strains tested. A once-daily dosage of 400 mg i.v. or 500 mg p.o. might be sufficient for treatment of urinary tract infections caused by highly susceptible pathogens. A twice-daily dosing scheme seems to be preferable for complicated infections caused by pathogens with intermediate susceptibilty (MIC≥1 μg/ml) or for empiric therapy.     

Molecular Characterization and Transfer Among Staphylococcus Strains of a Plasmid Conferring High-Level Resistance to Mupirocin

 In this work, mupirocin resistance was correlated with the presence of plasmids in methicillin-resistant Staphylococcus aureus (MRSA) strains isolated in the Rio de Janeiro Federal University Hospital in Brazil, where topical mupirocin has been used extensively since 1990. Of 19 strains studied, those exhibiting high-level resistance carried a large and relaxable plasmid of about 35 kb. Mupirocin-sensitive derivatives, obtained by growth at 42  °C of a strain exhibiting high-level resistance, were devoid of the large plasmid, which was designated pMG1. Mupirocin resistance was transferred to strain RN8411 during overnight filter-matings at low frequencies (7.0×10^–9/donor). The pMG1 plasmid was shown to be responsible for high-level mupirocin resistance in our isolates and to be incompatible with pGO1. Hybridization experiments suggested that mupirocin resistance in pMG1 is due to the presence of the ileS-2 gene. The pMG1 plasmid was successfully and bidirectionally transferred from Staphylococcus aureus to Staphylococcus epidermidis, suggesting that the latter may be a reservoir of this resistance plasmid. No transfer was detected to Staphylococcus haemolyticus. The development of self-transferable high-level mupirocin resistance should be considered when using mupirocin to control the spread of MRSA in hospitals.     

Evaluation of Ceftazidime Concentration Released in Agar from an E Test Strip

 The aim of this study was to evaluate, using high-performance liquid chromatography, the concentration of ceftazidime in agar released from an E test strip, sampling at the edge of the strip at different points (1, 2, 4, 8, 16, 32, 64, and 128 μg/ml) at 6, 15, and 24 h after its deposition on uninoculated plates. From 6 to 24 h, the ceftazidime concentration in agar increased at the graduations 1, 2, and 4 μg/ml (+140, +82, and +58%, respectively), remained fairly constant at 8 μg/ml (–1.9%), and decreased at 16, 32, 64, and 128 μg/ml (–25, –44, –36, and –58%, respectively). In the 6–24 h range, the ceftazidime concentrations between 16 and 1 μg/ml were ±1 serial dilution of the values reported on the strip, confirming the accuracy of the E test in agar.     

Comparative in vitro activity of cefpiramide,a new parenteral cephalosporin

The in vitro activity of cefpiramide was compared with that of nine other cephalosporins by determining the MIC values by means of an agar dilution method for 300 representative clinical isolates of non-fastidious bacteria. Cefpiramide had a broad-spectrum of activity, similar to that of the third-generation cephalosporins. As judged by the MIC50 and the MIC90 values, cefpiramide was one of the most active cephalosporins againstPseudomonas aeruginosa (MIC50 2.9 µg/ml, MIC90 64 µg/ml), staphylococci (MIC50 1.2 µg/ml, MIC90 10 µg/ml) andEnterococcus faecalis (MIC50 13 µg/ml, MIC90 43 µg/ml). Cefpiramide had moderate activity againstEnterobacteriaceae (MIC50 4 µg/ml, MIC90 108 µg/ml), comparable to that of the older second-generation cephalosporins. Ticarcillin-resistant strains of gram-negative rods were inhibited by higher concentrations of cefpiramide than ticarcillin-susceptible strains.     

Flucytosine Primary Resistance in Candida Species and Cryptococcus neoformans

 The in vitro activity of flucytosine (5FC) against 1,140 clinical isolates of Candida spp. and Cryptococcus neoformans was evaluated and compared with the activity of amphotericin B, fluconazole and itraconazole. Overall, 87.72% (1,000/1,140) of yeasts were susceptible to 5FC. This agent showed less potent in vitro activity against Candida glabrata, Candida krusei, Candida guilliermondii and Cryptococcus neoformans (MIC90s, 8–16 μg/ml) and intermediate activity or resistance to 6.5% of Candida albicans, 5.1% of Candida tropicalis and 0.8% of Candida parapsilosis strains. Amphotericin B showed potent activity against isolates with an MIC of 5FC≥8 μg/ml. A total of 112 of 140 strains that were 5FC-intermediate or -resistant showed decreased susceptibility to azoles (P<0.01).     

Validity of Screening Procedures for Glycopeptide-Resistant Enterococci

Screening agars containing different vancomycin concentrations and different susceptibility testing procedures were compared to determine their validity for the detection of glycopeptide-resistant enterococci (GRE). Direct streaking of rectal swabs over the surface of a commercially available agar (Enterococcosel; Becton Dickinson, Germany) containing 4 μg/ml and 16 μg/ml vancomycin was followed by incubation for 24 to 48 h. Susceptibility tests were done by microbroth dilution, disk diffusion, and the E test (AB Biodisk, Sweden). The microbroth dilution method according to National Committee for Clinical Laboratory Standards (NCCLS) was used as the gold standard for detection of GRE. Resistant and intermediately susceptible enterococcal isolates were differentiated to the species level. To detect resistance genes, the polymerase chain reaction was performed on all intermediately resistant isolates, on all isolates of VanB phenotype, and on 30% of isolates of VanA phenotype. Screening agar containing 4 μg/ml vancomycin displayed high sensitivity (97.6%) but only low specificity (35%) for the detection of GRE. Screening agar containing 16 μg/ml vancomycin had a high specificity of 89.3% and only a slightly lower sensitivity (92.7%) than the screening agar containing 4 μg/ml vancomycin. For the disk diffusion test, a breakpoint of <16 mm yielded the optimal combination of sensitivity (98.8%) and specificity (99.6%). Both sensitivity and specificity of the E test for GRE detection were 100%. However, the E test is too expensive for testing of all enterococci. In conclusion, the combination of an inexpensive screening agar with either the E test or the disk diffusion test constitutes a valid and cost-effective method for the detection of GRE from screening specimens.     

In vitro activity of temocillin against prevalent extended-spectrum beta-lactamases producing Enterobacteriaceae from Belgian intensive care units

Temocillin is a narrow spectrum penicillin with high stability to most β-lactamases including AmpC types and extended-spectrum types (ESBLs). We have analysed its in vitro activity against 652 clinical isolates of Enterobacteriaceae prospectively collected from patients hospitalised in intensive care units at seven different university hospitals in Belgium in 2005. Strains were screened for ESBL production using cefotaxime and ceftazidime screen agar plates and by double ESBL E-tests. The MIC of temocillin and of five comparators was determined using the E-test method. ESBLs were characterized at one central laboratory by isoelectric focusing, PCR for bla genes of the SHV, TEM, and CTX-M families, and by DNA sequencing. The prevalence of ESBL-producing Enterobacteriaceae averaged 11.8% and ranged between 3.0 and 29% in the different hospitals. Meropenem exhibited the highest in vitro activity overall (mode MIC 0.064 μg; MIC₉₀; 0.19 μg/ml), whereas ceftazidime (MIC₉₀ > 256 μg/ml) and ciprofloxacin (MIC₉₀ > 32 μg/ml) scored the worst. Temocillin was active against more than 90% of the isolates including most AmpC- and ESBL-producing isolates. These data indicate the well preserved activity of temocillin over the years against Enterobacteriaceae and show the wide variation in prevalence of ESBL-producing Enterobacteriaceae isolates in Belgian intensive care units. Prospective clinical studies are, however, needed to validate the usefulness of temocillin in the treatment of microbiologically documented infections caused by ESBL- and/or AmpC- overproducing nosocomial Enterobacteriaceae pathogens.     

In Vitro Susceptibility of Environmental Cryptococcus neoformans Variety neoformans Isolates from Turkey to Six Antifungal Agents, Including SCH56592 and Voriconazole

 The in vitro antifungal susceptibility of 27 environmental (pigeon droppings) isolates of Cryptococcus neoformans var. neoformans, isolated from throughout Turkey, to six antifungal agents (amphotericin B, flucytosine, fluconazole, voriconazole, itraconazole, and SCH56592) was studied. Voriconazole, itraconazole, and SCH56592 all showed comparable activity and were more active than the remaining three antifungal agents tested. Overall, SCH56592 was the most active agent (MIC90, 0.015 μg/ml, at both 48 and 72 h), followed by itraconazole (MIC90, 0.03 μg/ml, at both 48 and 72 h) and voriconazole (MIC90, 0.25 μg/ml, at both 48 and 72 h), respectively. Antifungal susceptibility data for environmental isolates may reflect patterns for the clinical isolates recovered from patients from the same geographic area.     

Antibacterial Activity of Moxifloxacin (Bay 12-8039) against Aerobic Clinical Isolates, and Provisional Criteria for Disk Susceptibility Tests

 Moxifloxacin (Bay 12–8039), ciprofloxacin, and levofloxacin were compared in vitro against 1074 clinical isolates gathered from different medical centers throughout North America during the winter months of 1997. Moxifloxacin E tests and broth microdilution tests gave comparable results. Moxifloxacin was particularly potent against respiratory pathogens such as Haemophilus influenzae and Streptococcus pneumoniae. Ciprofloxacin was the most potent study drug against the family of Enterobacteriaceae and Pseudomonas spp. For tests of 5 μg moxifloxacin disks, zone size criteria of ≤17 mm for resistant (MIC ≥8 μg/ml) and ≥21 mm for susceptible (MIC ≤2 μg/ml) are provisionally proposed for use while clinical trials are under way.     

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.     

Variations of agar screen tests for detection of methicillin resistance in staphylococci: focus on cefoxitin

Members of the genus Staphylococcus are among the most important human pathogens, and strains demonstrating resistance to methicillin are an increasing problem worldwide, both within and outside of hospital environments. The objective of this study was to evaluate the use of variations of agar screening tests with cefoxitin and oxacillin to detect methicillin resistance in staphylococcal isolates. The agar screening test with cefoxitin (4 μg/ml) showed 99.4% accuracy for detecting both S. aureus and coagulase-negative staphylococci. The performance of the agar screening test with cefoxitin (4 μg/ml) either equaled or was superior to the other agar screening test variations evaluated and can be used to characterize the presence of the mecA gene among staphylococcal species.     

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.     

Comparative In Vitro Activity of Gatifloxacin Against Stenotrophomonas maltophilia and Burkholderia Species Isolates Including Evaluation of Disk Diffusion and E Test Methods

 The in vitro activity of gatifloxacin, a new fluoroquinolone, was compared to that of five other fluoroquinolones against 105 Stenotrophomonas maltophilia isolates and 52 Burkholderia spp. isolates. The gatifloxacin MICs were determined using the broth microdilution method and the E test (AB Biodisk, Sweden); these methods were compared for test accuracy, and 5 μg disk zone diameters were compared for interpretive accuracy using the standardized disk diffusion method. In terms of potency, gatifloxacin was most similar to sparfloxacin and trovafloxacin against Stenotrophomonas maltophilia (MIC50, 0.5–1 mg/l) and Burkholderia spp. (MIC50, 1–2 mg/l). This activity was greater than that of ciprofloxacin, levofloxacin or ofloxacin (MIC50, ≥2 mg/l) against Stenotrophomonas maltophilia isolates but comparable to that of levofloxacin against the Burkholderia spp. (60% susceptible at ≤2 mg/l). The E test results compared well with the reference dilution test results (81–97% at ±1 log₂ dilution). The disk diffusion test using previously suggested breakpoints for other bacteria (≥18 mm or ≤2 mg/l for susceptible and ≤14 mm or ≥8 mg/l for resistant) also performed well, at >90% categorical agreement. The activity of gatifloxacin is comparable to that of other newer quinolones against isolates of Stenotrophomonas maltophilia and Burkholderia spp., and susceptibility testing using simple qualitative and quantitative methods appears to function well with these drug/organism combinations.     

Evaluation of discrepancies between oxacillin and cefoxitin susceptibility in coagulase-negative staphylococci

The Clinical and Laboratory Standards Institute (CLSI) recommends testing coagulase-negative staphylococci (CoNS) strains to determine resistance against oxacillin by testing for mecA, PBP2a, or with cefoxitin disk. However, discrepant results of resistance to oxacillin and susceptibility to cefoxitin were found. In this study, we aimed to investigate the oxacillin resistance and cefoxitin susceptibility of CoNS in Taiwan. Of 9,017 strains collected from 2005 to 2010, 131 (1.5%) of the isolates were oxacillin-resistant and cefoxitin-susceptible. Species identification was carried out using the Vitek 2 system or 16S ribosomal RNA sequencing. Oxacillin minimum inhibitory concentrations (MICs) were examined by the agar dilution method. The presence of mecA and the activity of β-lactamase were performed by polymerase chain reaction (PCR) and Cefinase disks, respectively. Overall, 33% (43/129) of the strains carried mecA and 43% (37/86) of mecA-negative isolates tested positive for β-lactamase. The remaining 49 isolates were negative for both mecA and β-lactamase, and were mainly Staphylococcus cohnii ssp. urealyticus and S. saprophyticus (oxacillin MICs 0.5–2 μg/ml) obtained from bloodstream and urinary tract infections. Our study suggests that incorrect reporting can be found in CoNS using cefoxitin disk alone to determine the susceptibility to oxacillin, and the strains should be further tested for oxacillin MICs and detection of the mecA gene or β-lactamase activity.     

Relative Potency of Telithromycin, Azithromycin and Erythromycin Against Recent Clinical Isolates of Gram-Positive Cocci

 A ketolide (telithromycin), an azalide (azithromycin) and a macrolide (erythromycin) were tested against 2,733 isolates of gram-positive cocci gathered from 11 different medical centers. Telithromycin was active against erythromycin-resistant staphylococci that were susceptible to clindamycin but was not active against those that were resistant to clindamycin. More than 99% of all Streptococcus pneumoniae and Streptococcus pyogenes isolates were susceptible to 1 μg/ml of telithromycin including erythromycin- and azithromycin-resistant strains. Telithromycin was not only more potent than azithromycin against macrolide-susceptible strains, it was also active against most macrolide-resistant strains. Although the prevalence of macrolide-resistant pneumococci increased from 19% to 27% between 1997 and 1999, macrolide resistance among other gram-positive cocci did not change substantially in that 2-year period.