Fatal disseminated Trichoderma longibrachiatum infection in an adult bone marrow transplant patient: species identification and review of the literature.

Trichoderma longibrachiatum was recovered from stool surveillance cultures and a perirectal ulcer biopsy specimen from a 29-year-old male who had received an allogeneic bone marrow transplant for acute lymphoblastic leukemia. The amphotericin B (2.0 microgram/ml) and itraconazole (1.0 microgram/ml) MICs for the organism were elevated. Therapy with these agents was unsuccessful, and the patient died on day 58 posttransplantation. At autopsy, histologic sections from the lungs, liver, brain, and intestinal wall showed infiltration by branching septate hyphae. Cultures were positive for Trichoderma longibrachiatum. While Trichoderma species have been recognized to be pathogenic in profoundly immunosuppressed hosts with increasing frequency, this is the first report of probable acquisition through the gastrointestinal tract. Salient features regarding the identification of molds in the Trichoderma longibrachiatum species aggregate are presented.

Antimicrobial activity and spectrum of LB20304, a novel fluoronaphthyridone.

Compound LB20304 is a fluoronaphthyridone carboxylic acid with a novel pyrrolidine substituent. This drug was compared with ciprofloxacin, levofloxacin, ofloxacin, and trovafloxacin against over 800 pathogens, most from blood stream infections, by National Committee for Clinical Laboratory Standards reference methods. LB20304 was the most active agent against gram-positive species including strains observed to be resistant to other fluoroquinolones and glycopeptides. The potency of LB20304 (MIC50, 0.03 micrograms/ml) against the Enterobacteriaceae was exceeded only by that of ciprofloxacin (0.015 micrograms/ml). It has limited activity against gram-negative anaerobes.

Avoparcin, a glycopeptide used in animal foods: antimicrobial spectrum and potency tested against human isolates from the United States.

Avoparcin is a glycopeptide antimicrobial that is widely used as a growth promoter in animal feeds in portions of Western Europe. Recently, concern has emerged about the possible contribution of avoparcin use to the emergence of glycopeptide resistance in enterococci. Relatively little information exists on the spectrum of activity and potency of avoparcin. We studied the activity of avoparcin compared to vancomycin, teicoplanin, and 3 other antimicrobials against 814 recent human clinical isolates, including Staphylococcus spp. (240 strains), Streptococcus spp. (90 strains), and Enterococcus spp. (60 strains), using reference susceptibility test methods. Our results indicate that avoparcin was less potent than either vancomycin or teichoplanin against staphylococci (MIC50, 4 micrograms/ mL). There was a good correlation of avoparcin MICs with the MICs for vancomycin and teichoplanin for most species; however, the avoparcin MICs for Enterococcus spp. of the vanB phenotype were quite variable (MIC range, 2 to > 16 micrograms/mL). For Staphylococcus haemolyticus, high avoparcin MICs (> or = 16 micrograms/mL) were associated with oxacillin resistance. These results are relevant to the debate concerning the appropriateness of continued use of avoparcin as a growth promoter in animal husbandry. In particular, avoparcin as a glycopeptide with limited potency against some staphylococci seems to represent a theoretically greater risk for selecting glycopeptide resistance among staphylococci, but may not represent any greater threat for the selection of resistance in enterococci.

Standardization of antifungal susceptibility testing.

The application of in-vitro antifungal susceptibility testing to clinical research and to the guidance of antifungal therapy has been limited by a lack of reproducibility and uncertain clinical relevance. As a result of studies of the identified variables impacting on in-vitro susceptibility results, the National Committee for Clinical Laboratory Standards have proposed a standardized antifungal susceptibility test method M27-P. The degree of intra- and inter-laboratory reproducibility which can be achieved with this method have been defined in multi-laboratory collaborative studies. More convenient methods (microdilution broth and stable gradient technology) have been evaluated relative to the proposed standard method and the potential for a similar process with a disc diffusion method is apparent. A modification of this standard method for susceptibility testing of filamentous fungi appears promising. The existence of a standardized method and of alternative methods with a defined relationship to the proposed standard, facilitates meaningful analysis of published studies addressing the issue of clinical relevance of antifungal susceptibility testing. As a result of this process, correlation of MICs determined in vitro with clinical response to therapy is beginning to emerge, most notably in relation to fluconazole therapy for oropharyngeal candidosis associated with infection with the human immunodeficiency virus.

A Mycobacterium malmoense-specific DNA probe from the 16S/23S rRNA intergenic spacer region.

Mycobacterium malmoense was first described in 1977. It is now recognized as an opportunistic human pathogen which can be difficult to identify using standard methods. M. malmoense may be underestimated as the causative agent of clinical disease because of the recognized difficulties in its primary cultivation and identification. In this study, the nucleotide sequence of the 16S/23S rRNA intergenic spacer region from five clinical isolates of M. malmoense has been determined, in order to develop a PCR-based DNA probe assay to facilitate the early identification of this organism. The DNA sequence generated was utilized to design an oligunucleotide probe that specifically hybridizes with M. malmoense. The ability of this DNA probe to detect geographically distinct M. malmoense isolates was investigated. The value of this DNA probe was realized by its ability to differentiate three isolates of the Mycobacterium avium complex, which had been misidentified as M. malmoense using conventional biochemical methods.

Detection of extended-spectrum beta-lactamase (ESBL)-producing strains by the Etest ESBL screen.

Resistance to contemporary broad-spectrum beta-lactams, mediated by extended-spectrum beta-lactamase (ESBL) enzymes, is an increasing problem worldwide. The Etest (AB Biodisk, Solna, Sweden) ESBL screen uses stable gradient technology to evaluate the MIC of ceftazidime alone compared with the MIC of ceftazidime with clavulanic acid (2 micrograms/ml) to facilitate the recognition of strains expressing inhibitable enzymes. In the present study, ESBL-producing strains (17 Escherichia coli transconjugants) were studied to define "sensitive" interpretive criteria for the Etest ESBL screen. These criteria (reduction of the ceftazidime MIC by > 2 log2 dilution steps in the presence of clavulanic acid) defined a group of 92 probable ESBL-positive organisms among the 225 tested strains of Klebsiella species and E. coli having suspicious antibiogram phenotypes. With a subset of 82 clinical strains, the Etest ESBL screen was more sensitive (100%) than the disk approximation test (87%) and was more convenient. The MICs of ciprofloxacin, gentamicin, and tobramycin at which 50% of isolates are inhibited were 16- to 128-fold higher (coresistance) for the ESBL screen-positive group of strains than for the ESBL screen-negative group of strains. Some strains for which cephalosporin MICs were elevated and which were Etest ESBL screen negative were also cefoxitin resistant, i.e., consistent with a chromosomally mediated AmpC resistance phenotype. The Etest ESBL screen test with the ceftazidime substrate appears to be a useful method for detecting or validating the presence of enteric bacilli potentially producing this type of beta-lactamase.

Role of the microbiology laboratory in monitoring and identifying resistance: use of molecular biology.

Antimicrobial resistance is an increasing problem in the United States. Early detection of emerging trends in antimicrobial resistance may facilitate implementation of effective control measures. Most antimicrobial susceptibility testing is qualitative, in that it categorizes isolates as susceptible, intermediate, or resistant. This approach is relatively inexpensive and generally adequate for clinical purposes. Qualitative susceptibility testing has some limitations for monitoring for emerging resistance. Selective quantitative susceptibility testing may be useful in detecting early trends toward elevated minimal inhibitory concentrations. Molecular methods have a role also in characterizing mechanisms of resistance and in the typing of resistant strains to determine patterns of spread. Laboratory monitoring of emerging resistance must be associated with an effective infection control policy and a willingness to modify practice in a rational manner based upon the trends detected.

Clindamycin resistance among erythromycin-resistant Streptococcus pneumoniae.

The increasing proportion of Streptococcus pneumoniae isolates with reduced susceptibility to penicillin has created an urgent need for therapeutic alternatives to some beta-lactam agents. Clindamycin is an antimicrobial agent with excellent bioavailability after oral administration which has been considered for the therapy of community-acquired pneumococcal otitis media. Using the Etest methodology, we have studied the in vitro susceptibility of 59 erythromycin-resistant strains of S. pneumoniae to clindamycin, penicillin, trimethoprim-sulfamethoxazole, and rifampin. The study also addressed the impact of the susceptibility test medium [Mueller-Hinton (MH) vs IsoSensitest (Iso), both 5% blood supplement] on the results. A total of 20 isolates (37%) displayed constitutive clindamycin resistance on Iso blood agar, compared with only 11 (22%) on MH blood agar. The remaining nine strains found to be clindamycin susceptible on MH manifested resistance only with erythromycin induction. Resistance to penicillin, rifampin, and trimethoprim-sulfamethoxazole in erythromycin-resistant isolates was 83%, 2%, and 85%-89% (medium dependent), respectively. These results indicate that the choice of susceptibility test medium affects the expression (constitutive or inducible) of macrolide-lincosamide-streptogramin (MLS) resistance in S. pneumoniae. In addition, the common assumption that erythromycin resistance in S. pneumoniae implies clindamycin resistance may need to be reconsidered and routine susceptibility tests (including induction if MH medium is used) should be considered for MLS-class drugs.

Phenotypic detection of mec A-positive staphylococcal blood stream isolates: high accuracy of simple disk diffusion tests.

Detection of oxacillin-resistance in staphylococci by phenotypic methods remains problematic. Although standardized susceptibility test methods are adequate for Staphylococcus aureus, many are less satisfactory for the coagulase-negative staphylococci (CNS). We have studied 108 consecutive blood culture isolates of staphylococci. The mec A gene was detected by PCR in one S. aureus and 55 CNS isolates. Susceptibility testing was performed as follows: oxacillin (1-microgram), ceftizoxime (30-microgram), and cephalothin (30-microgram) by disk diffusion; oxacillin, ceftizoxime, cephalothin, methicillin, ampicillin, ampicillin/ sulbactam, penicillin, cefazolin, imipenem, and meropenem by the broth microdilution method. In addition, isolates were tested by the oxacillin agar screen plate method. The single oxacillin-resistant S. aureus strain was detected by all oxacillin susceptibility test methods and by the ceftizoxime disk and MIC methods. Two oxacillin-susceptible S. aureus were intermediate (minor error) by ceftizoxime broth microdilution (MIC, 16 micrograms/mL). The most sensitive, simple phenotypic methods for detection of oxacillin-resistant CNS (mec A positive) were as follows: oxacillin disk diffusion at 98%, oxacillin screen plate at 91%, oxacillin broth microdilution at 87%, ceftizoxime disk diffusion at 100%, ceftizoxime broth microdilution at 87%, and methicillin broth microdilution at 83%. These results indicate that oxacillin and ceftizoxime disk diffusion tests are the most accurate phenotypic methods in routine clinical use for detection of oxacillin-resistant CNS. Oxacillin broth microdilution MIC testing (2% NaCl supplement) would perform more satisfactorily (100% sensitivity) with an adjusted interpretive breakpoint at < or = 0.5 microgram/mL, in contrast to the lower accuracy of the "so-called" reference agar screen test.

DNA macrorestriction profiles and antifungal susceptibility of Candida (Torulopsis) glabrata.

Candida (Torulopsis) glabrata is an emerging nosocomial pathogen that may be relatively resistant to fluconazole. A series of 75 isolates (blood, urine, tissue, and other sites) from 16 patients (1 to 12 isolates per patient) at a single university medical center were analyzed by pulsed field gel electrophoresis (PFGE) of restriction endonuclease digests of chromosomal DNA. The MICs of the isolates for amphotericin B, flucytosine, fluconazole, and itraconazole, were determined by a microdilution broth method. A preliminary study of seven restriction enzymes, three producing small fragments (Hinf I, Hind III, Eco RI) and four producing large fragments (Eag I, BssH II, Sfi I, Not I) identified Not I as giving interpretable banding patterns. Isolates were considered of different types if they differed by two or more bands. Sixteen distinct DNA types (A to P) were identified. Karyotyping was used an an additional technique to compare strains with a common PFGE type. Most patients were colonized or infected with a single type at multiple body sites and over time. One PFGE type was shared by four patients housed in different areas of the institution at different times. For two of these four patients, the karyotype was also indistintuishable. Five patients were each colonized with two distinct types. The MIC of the strains studied were amphotericin B 0.5-1.0 microgram/ml (MIC90 = 1.0 microgram/ ml), 5-fluorocytosine 0.25-->256 micrograms/ml (MIC90 = 2 micrograms/ml), fluconazole 0.25-->128 micrograms/ml (MIC90 = 32 micrograms/ml), and itraconazole 0.06-8.0 micrograms/ml). Molecular typing by PFGE using Not I digestion is a useful technique for epidemiological investigation as epidemiologically related isolates are generally identical and epidemiologically unrelated isolates are different by this method.

Multi-center validation of proposed disk diffusion susceptibility testing interpretive criteria for lomefloxacin using more than 1,500 clinical isolates.

The accuracy of disk diffusion susceptibility testing of lomefloxacin was evaluated using 1,555 recent clinical isolates from ten medical centers. The isolates were rapidly growing nonfastidious aerobic species (1,501 isolates) and Haemophilus species (54 isolates), each found as an indicated species in the product package insert. Applying the recently proposed modification of disk diffusion interpretive criteria (susceptible at > or = 20 mm and resistant at < or = 16 mm), absolute categorical agreement for nonfastidious aerobes (1,501 strains) was 95.5% with 0.5% very major and 0.1% major errors (error rates calculated using all tested strains as the denominator). The intermethod discord (MIC vs disk diffusion) was 0.5%. This contrasts to the current NCCLS recommended criteria (susceptible at > or = 22 mm and resistant at < or = 18 mm) where the absolute categorical agreement was significantly less (89.6%) with 0.2% very major, and 0.3% major errors, and the intermethod discord was 7.1%. For Haemophilus species (54 strains), intermethod agreement was complete using either the current NCCLS interpretive criteria or the modified criteria. These multicenter (ten laboratories) data support the acceptance of the proposed modification of disk diffusion interpretive criteria for 10-microgram lomefloxacin disks.

Susceptibility testing interpretive criteria for levofloxacin when testing respiratory pathogens, Haemophilus influenzae and Moraxella catarrhalis.

The more active L-isomer, levofloxacin, of the racemic ofloxacin mixture has been under development for therapeutic use. In this study, we evaluated the activity of ofloxacin, levofloxacin, and D-ofloxacin against the fastidious respiratory tract pathogens Haemophilus influenzae and Moraxella catarrhalis. Levofloxacin was two-fold more active than ofloxacin against H. influenzae (MIC90, 0.015 microgram/ml), and D-ofloxacin was least active (MIC90, 1 microgram/ml). For M. catarrhalis the MIC90 values were 0.03 microgram/ml, 0.06 microgram/ml, and 2 micrograms/ml for levofloxacin, ofloxacin, and D-ofloxacin, respectively. For disk diffusion susceptibility testing, Chocolate Mueller-Hinton agar (CMH) was considered preferable to Haemophilus test medium (HTM) because it supported the growth of all of 105 H. influenzae strains whereas five strains failed to grow on HTM. In addition, the margins of the zones of inhibition were more distinct on CMH and the Haemophilus species strains with elevated fluoroquinolone MICs were readily distinguished. The superior growth on CMH was reflected in a reduction of inhibition zone diameters of 2-3 mm relative to the inhibition zone diameters on HTM. The previously proposed interpretive criteria for the 5 microgram disk diffusion susceptibility test (susceptible at > or = 17 mm) results in complete categorical agreement with the reference microdilution broth method for M. catarrhalis on Mueller Hinton agar and for H. influenzae on HTM and CMH. However, the minimum diameter of the zone of inhibition recorded for a member of the dominant population of either species was considerably greater (25 mm) than 17 mm on any of the media tested.

Emerging resistance to antimicrobial agents in gram-positive bacteria. Enterococci, staphylococci and nonpneumococcal streptococci.

Staphylococci (Staphylococcus aureus and coagulase-negative Staphylococcus species) and enterococci are the aetiological organisms in 47 to 52% of nosocomial blood stream infections and approximately 30% of all nosocomial infections in the US. In European intensive care units, almost half of all infections are attributed to staphylococci. The streptococci have also become increasingly important because of the modified virulence of Streptococcus pyogenes strains, and the emerging role of the viridans group streptococci as a cause of potentially fatal bacteraemia in the neutropenic host. Resistance to available antimicrobial agents is increasing and includes, in particular, resistance to the glycopeptides (vancomycin and teicoplanin) amongst enterococci, resistance to penicillinase-resistant penicillins (oxacillin and methicillin) and fluoroquinolones (ciprofloxacin and ofloxacin) amongst staphylococci, and resistance to penicillin and some other beta-lactams amongst viridans group streptococci. New compounds for effective therapy of infection with antimicrobial-resistant Gram-positive species are needed urgently. To this end, the streptogramin combinations [quinupristin/dalfopristin (RP 59500; Synercid)], everninomycin derivatives (SCH 27899), oxazolidinones (U-100572, U-100766) and several newer fluoroquinolones (clinafloxacin, DU 6859a, grepafloxacin, levofloxacin, sparfloxacin, trovafloxacin) are under rapid development and clinical investigation.

Preliminary interpretive criteria for disk diffusion susceptibility testing of SCH 27899, a compound in the everninomicin class of antimicrobial agents.

As antimicrobial resistance among Gram-positive species becomes more common, alternative agents need to be developed for the therapy of serious infections. SCH 27899 is a compound from the everninomicin class of antimicrobial agents that possesses a potent Gram-positive spectrum. We evaluated three disk concentrations (0.25, 1, and 5 micrograms) of three SCH 27899 formulations including SCH 27899 base (SCHB), N-methylglucamine SCH 27899 (NMG-SCH), and NMG-SCH complexed with hydroxypropyl beta-cyclodextrin. Disk zone diameters were correlated with minimum inhibitory concentration for 209 aerobic, nonfastidious Gram-positive strains and selected Gram-negative bacilli to develop disk diffusion interpretive criteria. No significant differences in activity were noted among the three SCH 27899 preparations. Of the three disk concentrations, the correlation coefficient was greatest (r = 0.88) for the 5-micrograms SCHB disk test. For a tentative break point of < or = 2 micrograms SCHB/ml, preliminary disk interpretive criteria were: susceptible at > or = 12 mm, intermediate at 10-11 mm, and resistant at < or = 9 mm (absolute categorical agreement, 99.5%). Zones were small secondary to drug solubility and diffusion limitations. Using these criteria for the SCHB 5-micrograms disks, nearly all of the tested Gram-positive organisms were susceptible including methicillin-resistant staphylococci and vancomycin-resistant enterococci.

Comparative antimicrobial activity and spectrum of CP-99,219, a novel fluoroquinolone, tested against ciprofloxacin-resistant clinical isolates.

The fluoroquinolones have an established role in treatment of infection with aerobic gram negative rods. The increased importance of gram positive nosocomial infection and of acquired fluoroquinolone resistance has stimulated a search for new compounds with enhanced potency and spectrum. CP-99,219 is a novel compound in this class with enhanced activity against gram positive organisms. We have studied the activity of CP-99,219 relative to ciprofloxacin, fleroxacin, ofloxacin, and sparfloxacin using test panels of organisms with a high proportion of ciprofloxacin resistance. CP-99,219 is more potent than any of the other four compounds against both gram positive and gram negative bacteria. The activity of CP-99,219 against many bacteria resistant to the established agents, warrants further in vitro and clinical studies.

Cross-resistance analysis for clinafloxacin compared with ciprofloxacin, fleroxacin, ofloxacin, and sparfloxacin using a predictor panel of ciprofloxacin-resistant bacteria.

Clinafloxacin was significantly more active against fluoroquinolone-resistant organisms than ciprofloxacin, ofloxacin, sparfloxacin and fleroxacin. Clinafloxacin inhibited 65% of isolates at the recommended breakpoint (< or = 1 mg/L) compared with only 30.0% for ciprofloxacin, 31.7% for ofloxacin, 32% for fleroxacin, and 37.7% for sparfloxacin at their recommended breakpoints. No strain susceptible to ciprofloxacin was resistant to the other compounds tested.