Recovery of Coccidioides immitis from blood and abscess fluid using the BacT/alert system.

We report the recovery of Coccidioides immitis from the blood and abscess fluid of two separate patients by using two automated blood culture systems. In the first case, an aspirate from a neck abscess containing C. immitis spherules was serially diluted and inoculated into liquid media used by the BacT/Alert and the Bactec NR660 blood culture systems. BacT/Alert bottles inoculated with 10(5), 10(4), 10(3), 10(2), 10 and two spherules produced a positive signal at 19, 24, 35, 42, 57, and 62 h postinoculation, respectively. Bactec NR660 bottles containing > 10(2) shperules and 10 spherules produced a positive signal after approximately 72 and 96 h of incubation, respectively. In the second case, a blood specimen incubated in BacT/Alert blood culture both was signaled positive after 82 h of incubation. No organisms were detected by Gram stain of the broth, but C. immitis grew after blind subculture. Our observations demonstrate that these rapid blood culture systems are capable of supporting growth of C. immitis. To our knowledge, this report is the first to detect C. immitis by these blood culture systems.

Cultivation of Nocardia spp. on chemically defined media for selective recovery of isolates from clinical specimens.

Isolation of Nocardia spp. from clinical specimens can be enhanced by the use of paraffin baiting, which relies on the selective ability of the organism to metabolize paraffin. We evaluated 44 Nocardia isolates, 18 group IV mycobacterial isolates, and 4 Streptomyces isolates for growth on blood agar (BA) and on carbon-free agar containing single or double substrates as follows: paraffin agar (PA), gelatin agar (GA), urea agar (UA), PA-gelatin (PG), and PA-urea (PU). The growth rates of Nocardia spp. on BA, PA, PU, and PG were similar; but 3-day-old colonies were larger on BA for 20 (45%) isolates. After longer incubations (7 to 14 days), some Nocardia colonies were larger on PA, PG and PU than they were on BA. Despite variable morphologies on BA, colonies on PA, PG, and PU were consistently smooth, creamy, and raised. Compared with growth on BA, the growth of mycobacteria was much slower on PA, PG, and PU, with poor growth on UA and GA. The growth of Streptomyces spp. was greatly enhanced on GA, PG, UA, and PU and was poorest on PA. Twelve sputum specimens seeded with Nocardia asteroides (10(4) CFU/ml) were inoculated onto BA and all chemically defined media. Nocardiae were recovered from 6 to 12 specimens grown on BA, GA, and UA; 11 of 12 specimens grown on PG; and 12 of 12 specimens grown on PA and PU. Only PA was able to suppress the growth of other microorganisms that were present in sputum specimens. These results suggest that chemically defined media containing PA may be useful for the selective isolation of Nocardia spp. from contaminated clinical specimens.

Large-scale use of polymerase chain reaction for detection of Mycobacterium tuberculosis in a routine mycobacteriology laboratory.

We investigated the use of DNA amplification by the polymerase chain reaction reaction (PCR) for detection of Mycobacterium tuberculosis from clinical specimens. Two-thirds of each sample was processed for smear and culture by standard methods, and one-third was submitted for DNA extraction, amplification of a 317-bp segment within the insertion element IS6110, and detection by agarose gel electrophoresis, hybridization, or both. DNA was prepared from over 5,000 samples, with 623 samples being culture positive for acid-fast bacilli. Of 218 specimens that were identified as M. tuberculosis, 181 (85%) were positive by PCR. In the M. tuberculosis culture-positive group, PCR was positive for 136 of 145 (94%) and 45 of 73 (62%) of the fluorochrome smear-positive and -negative specimens, respectively. Of 948 specimens that were either culture positive for mycobacteria other than M. tuberculosis or culture negative, 937 specimens were negative by PCR and 11 (1%) specimens initially appeared to be false positive for M. tuberculosis. The reason for discrepant results varied; some errors were traced to the presence of an inhibitor in the specimen (7.3% in unselected specimens), nucleic acid contamination, low numbers of organisms in the specimen antituberculosis therapy, and possible low-level nonspecific hybridization. In comparison with culture, the sensitivity, specificity, and positive predictive value were 83.5, 99.0, and 94.2%, respectively, for PCR. When PCR was corrected for DNA contamination, the presence of inhibitor, and culture-negative disease, the values became 86.1, 99.7, and 98.4%, respectively. If the results for multiple specimens submitted from the same patient are considered, no patient who had three of more sputum specimens tested would have been misdiagnosed.

Detection of Mycobacterium tuberculosis in clinical samples by two-step polymerase chain reaction and nonisotopic hybridization methods.

Detection of Mycobacterium tuberculosis in clinical specimens by the polymerase chain reaction (PCR) was compared with detection by culture. A 317-bp segment within the M. tuberculosis-specific insertion sequence IS6110 was amplified. The detection limit of the PCR assay for cultured mycobacteria was 50 cells per reaction by ethidium bromide-stained agarose gel electrophoresis and 5 cells per reaction by hybridization with an oligonucleotide probe conjugated with either digoxigenin or alkaline phosphatase (AP). This sensitivity was reduced fivefold in sputum specimens seeded with M. tuberculosis. Seventy-six clinical specimens were amplified and examined by the three detection methods. Both the digoxigenin and AP procedures were found to be more sensitive than agarose gel electrophoresis, but they were occasionally associated with a high background. An additional 308 specimens were examined only by agarose gel electrophoresis and the AP procedure. Of 71 specimens found to contain M. tuberculosis, amplified products were detected from 56 (79%) samples by agarose gel electrophoresis and/or the AP procedure. Of the additional 313 specimens that were culture negative for M. tuberculosis, 19 (6%) had amplified products detectable by agarose gel electrophoresis and/or the AP procedure. Compared with culture, PCR showed sensitivities and specificities of 55 and 98%, respectively, for agarose gel electrophoresis and 74 and 95%, respectively, for the AP procedure. Despite this low sensitivity, a rapid positive PCR result was accurate and clinically useful.

Agrobacterium tumefaciens peritonitis mimicking tuberculosis.

Agrobacterium species have been previously implicated in the development of clinical disease. We report what we believe to be the first case of ascites caused by Agrobacterium tumefaciens in a cirrhotic patient. Since the correct diagnosis was made only after laparoscopy-guided collection of specimens from two different tissues, we suggest that Agrobacterium may be an underdiagnosed pathogen in clinical situations in which tuberculosis is considered to be the cause of high-protein ascites.

Enhanced recovery of cytomegalovirus in conventional tube cultures with a spin-amplified adsorption.

Low-speed centrifugation-mediated adsorption was evaluated as an enhancement of infectivity of clinical and laboratory strains of cytomegalovirus (CMV) occurring with cells grown in conventional culture tubes. The time required for reporting of primary isolates of CMV from urine specimens adsorbed onto monolayers of WI-38 cells in culture tubes was calculated. Of 668 specimens adsorbed by the stationary phase (SP) method, 98 were positive by cytopathic effect (CPE) that required an average of 16.8 days for recovery in culture. However, the appearance of CPE required a shorter average time of 11.9 days for 70 CMV strains isolated from 283 specimens adsorbed in tube cultures by the spin-amplified (SA) method. In another phase of clinical CMV recovery, urine specimens were adsorbed by the SA method onto cell cultures grown in both shell vials and test tubes. Of 594 specimens inoculated, a total of 74 were positive by either CPE in test tubes or immunostaining-localized early antigen in shell vials. Approximately one-third of these CMV isolates were recovered only by CPE from specimens adsorbed by the SA method in test-tube cultures. In a related study to further evaluate differences between adsorption methods, the AD-169 laboratory strain of CMV was adsorbed by SP and SA methods onto MRC-5 cells grown in both culture vessels. Early antigen detection by immunomicroscopy was found in the infected cells at least 2 to 4 days prior to the appearance of CPE, regardless of adsorption procedure. In both vessels, the replication of AD-169 virus in cultures adsorbed by the SA method consistently exceeded that of virus adsorbed by the SP procedure. CPE occurred 24 to 48 h earlier and progressed two to four times more extensively; early antigen was expressed two- to fourfold greater within 24 to 48 h postinfection; and foci of infected cells containing late antigen were two to four times greater in number at 1, 2, and 5 days postinfection. Overall, the replication and enhancement of infectivity of laboratory and clinical strains of CMV as determined by CPE and early and late antigen expression occurred most efficiently with specimens adsorbed by the SA method onto cultures grown in conventional tubes or shell vials.

Diagnosis of intestinal tuberculosis by polymerase chain reaction on endoscopic biopsy specimens.

It is often difficult to establish the diagnosis of intestinal tuberculosis because of close similarities with other conditions, in particular, Crohn's disease. In the present study, we used the polymerase chain reaction (PCR) assay on endoscopic biopsy specimens obtained from a patient with chronic diarrhea. Positive hybridization was obtained with the Mycobacterium tuberculosis probe and the patient was treated with anti-tuberculous drugs with complete resolution of the endoscopic abnormalities. This study demonstrates that polymerase chain reaction assay can be used on endoscopic biopsy specimens to diagnose intestinal tuberculosis.

Rapid screening of natural products for antimycobacterial activity by using luciferase-expressing strains of Mycobacterium bovis BCG and Mycobacterium intracellulare.

The object of this study was to investigate the ability of a rapid luciferase assay to detect antimycobacterial activity in plant extracts. Recombinant strains of Mycobacterium bovis BCG (rBCG) and Mycobacterium intracellulare expressing firefly luciferase were used as the test organisms. Assays were conducted in a 96-well minitube format under biosafety level 2 conditions. Control and test wells were sampled immediately after inoculation and after 3 (recombinant M. intracellulare) and 5 (rBCG) days of incubation to measure luminescence with a microplate luminometer, and the relative change in luminescence was calculated as a percentage of control values. As an alternative test method, Alamar blue was added after 12 days of incubation, and changes in color were read visually. A total of 480 extracts were tested. Sixteen extracts were active against rBCG, and of those, seven were also active against recombinant M. intracellulare. With activity defined as a relative change in luminescence of < or = 1% (i.e., > or = 99% inhibition) and a persistence of blue color after addition of Alamar blue, there was 99.0% agreement between the two methods. Our results suggest that the luciferase assay is rapid and accurate and has the potential to greatly accelerate the evaluation of antimycobacterial activity in plant extracts in vitro. With this method, it is possible to screen a large number of samples in a short period of time.

Characterization of two unusual clinically significant Francisella strains.

We have isolated two phenotypically distinct nonfastidious Francisella strains (Fx1 and Fx2) from the blood of compromised patients with pneumonia and compared them with eight other Francisella strains, including Francisella tularensis biovar tularensis, F. tularensis biovar novicida, and F. philomiragia. Our isolates grew well on sheep blood agar, chocolate agar, modified Thayer-Martin agar, and Trypticase soy agar. Fx1 and Fx2 were determined to be within the Francisella genus by cellular fatty acid analysis and by the utilization of glucose, production of H2S and catalase, and lack of motility, oxidase, nitrate reductase, and gelatinase. They were additionally shown to belong to the species F. tularensis by sequencing of two variable regions comprising approximately 500 nucleotides of the 16S rRNA gene. Also, RNA probe hybridization confirmed their belonging to the species F. tularensis. However, the new strains, which are not identical, are distinguished from other F. tularensis strains by growth characteristics, repetitive extragenic palindromic PCR fragment pattern, and some biochemical tests. Key biochemical differences included the findings that Fx1 was positive for beta-galactosidase and arabinose hydrolysis and that both strains were citrulline ureidase positive and glycerol negative. Commercial F. tularensis antiserum agglutinated stock F. tularensis strains but not Fx1, Fx2, F. tularensis biovar novicida, or F. philomiragia; serum from either patient failed to agglutinate or only weakly agglutinated commercial antigen but showed agglutination when tested against each patient's respective isolate. Fx1 and Fx2 produced beta-lactamase. Because of their good growth, negative serology, and biochemical profile, the organisms could be misidentified in the clinical laboratory if standard strategies or commercial identification systems are used.

Luciferase in vivo expression technology: use of recombinant mycobacterial reporter strains to evaluate antimycobacterial activity in mice.

The development of new drugs and vaccines directed against Mycobacterium tuberculosis is severely impeded by the slow growth of this organism and the need to work under stringent biosafety conditions. These difficulties pose considerable obstacles when animal studies with M. tuberculosis are performed. We investigated whether a novel approach termed luciferase in vivo expression, using an enhanced luciferase-expressing mycobacterial strain, could be used to evaluate antimycobacterial activity in mice. Vectors that expressed firefly luciferase (lux gene) at high levels in the bacillus Calmette-Gu-erin (BCG) strain of Mycobacterium bovis were constructed for use in vivo. One recombinant BCG reporter strain (rBCG-lux) was selected for high-level expression of the lux gene product and for its ability to replicate in mice. Methodology to monitor in vivo growth of the rBCG-lux reporter strain in mice by direct assay of luciferase luminescence in organ homogenates was developed. The utility of this approach for assessing the in vivo efficacies of antimycobacterial compounds was evaluated. The activities of standard antimycobacterial drugs were directly apparent in mice infected with the rBCG-lux reporter strain by statistically significant reductions in spleen luminescence. In addition, antimycobacterial immunity was also evident in BCG-immunized mice, in which suppression of rBCG-lux growth in comparison with that in naive mice was clearly observed. The use of luciferase in vivo expression for the in vivo evaluation of antimycobacterial activity compared favorably with standard CFU determinations in terms of time, labor, expense, and statistical significance but permitted the evaluation of antimycobacterial drugs and immunity in mice in 7 days or less. Thus, the use of this technology can greatly accelerate the process of evaluation of antibiotics and immunogens in animal models for the slowly growing pathogenic mycobacteria.

Activities of tobramycin and six other antibiotics against Pseudomonas aeruginosa isolates from patients with cystic fibrosis.

The in vitro activity of tobramycin was compared with those of six other antimicrobial agents against 1,240 Pseudomonas aeruginosa isolates collected from 508 patients with cystic fibrosis during pretreatment visits as part of the phase III clinical trials of tobramycin solution for inhalation. The tobramycin MIC at which 50% of isolates are inhibited (MIC(50)) and MIC(90) were 1 and 8 microg/ml, respectively. Tobramycin was the most active drug tested and also showed good activity against isolates resistant to multiple antibiotics. The isolates were less frequently resistant to tobramycin (5.4%) than to ceftazidime (11.1%), aztreonam (11.9%), amikacin (13.1%), ticarcillin (16.7%), gentamicin (19.3%), or ciprofloxacin (20.7%). For all antibiotics tested, nonmucoid isolates were more resistant than mucoid isolates. Of 56 isolates for which the tobramycin MIC was > or = 16 microg/ml and that were investigated for resistance mechanisms, only 7 (12.5%) were shown to possess known aminoglycoside-modifying enzymes; the remaining were presumably resistant by an incompletely understood mechanism often referred to as "impermeability."

Aminoglycoside-resistance mechanisms for cystic fibrosis Pseudomonas aeruginosa isolates are unchanged by long-term, intermittent, inhaled tobramycin treatment.

Aminoglycoside-resistance mechanisms were characterized in Pseudomonas aeruginosa isolates from cystic fibrosis (CF) patients during a recent clinical trial of inhaled tobramycin. Impermeability, in which bacteria have reduced susceptibility to all aminoglycosides, was the predominant mode of resistance in isolates obtained both before and after 6 months of cyclic treatment with tobramycin or placebo administered by aerosol. Enzymatic resistance mechanisms were found in fewer than 10% of resistant isolates. P. aeruginosa from individual patients could be grouped on the basis of genetic relatedness. When enzymatic resistance was involved, all isolates in a group had elevated tobramycin MICs. When impermeability occurred, MICs of a genotypic group varied from susceptible to resistant. These findings suggest that impermeability resistance occurs in only a fraction of the P. aeruginosa population in lungs of persons with CF and that this form of resistance arises by a process involving multiple small changes in MIC.

Effect of chronic intermittent administration of inhaled tobramycin on respiratory microbial flora in patients with cystic fibrosis.

Pseudomonas aeruginosa endobronchial infection causes significant morbidity and mortality among cystic fibrosis patients. Microbiology results from two multicenter, double-blind, placebo-controlled trials of inhaled tobramycin in cystic fibrosis were monitored for longitudinal changes in sputum microbial flora, antibiotic susceptibility, and selection of P. aeruginosa isolates with decreased tobramycin susceptibility. Clinical response was examined to determine whether current susceptibility standards are applicable to aerosolized administration. Treatment with inhaled tobramycin did not increase isolation of Burkholderia cepacia, Stenotrophomonas maltophilia, or Alcaligenes xylosoxidans; however, isolation of Candida albicans and Aspergillus species did increase. Although P. aeruginosa tobramycin susceptibility decreased in the tobramycin group compared with that in the placebo group, there was no evidence of selection for the most resistant isolates to become most prevalent. The definition of resistance for parenteral administration does not apply to inhaled tobramycin: too few patients had P. aeruginosa with a tobramycin MIC >/=16 microgram/mL to define a new break point on the basis of clinical response.

Characterization of a Pseudomonas aeruginosa efflux pump contributing to aminoglycoside impermeability.

Pseudomonas aeruginosa can employ many distinct mechanisms of resistance to aminoglycoside antibiotics; however, in cystic fibrosis patients, more than 90% of aminoglycoside-resistant P. aeruginosa isolates are of the impermeability phenotype. The precise molecular mechanisms that produce aminoglycoside impermeability-type resistance are yet to be elucidated. A subtractive hybridization technique was used to reveal gene expression differences between PAO1 and isogenic, spontaneous aminoglycoside-resistant mutants of the impermeability phenotype. Among the many genes found to be up-regulated in these laboratory mutants were the amrAB genes encoding a recently discovered efflux system. The amrAB genes appear to be the same as the recently described mexXY genes; however, the resistance profile that we see in P. aeruginosa is very different from that described for Escherichia coli with mexXY. Direct evidence for AmrAB involvement in aminoglycoside resistance was provided by the deletion of amrB in the PAO1-derived laboratory mutant, which resulted in the restoration of aminoglycoside sensitivity to a level nearly identical to that of the parent strain. Furthermore, transcription of the amrAB genes was shown to be up-regulated in P. aeruginosa clinical isolates displaying the impermeability phenotype compared to a genotypically matched sensitive clinical isolate from the same patient. This suggests the possibility that AmrAB-mediated efflux is a clinically relevant mechanism of aminoglycoside resistance. Although it is unlikely that hyperexpression of AmrAB is the sole mechanism conferring the impermeability phenotype, we believe that the Amr efflux system can contribute to a complex interaction of molecular events resulting in the aminoglycoside impermeability-type resistance phenotype.

Antimicrobial effects of novel siderophores linked to beta-lactam antibiotics.

As a strategy to increase the penetration of antibiotic drugs through the outer membrane of gram-negative pathogens, facilitated transport through siderophore receptors has been frequently exploited. Hydroxamic acids, catechols, or very close isosteres of catechols, which are mimics of naturally occurring siderophores, have been used successfully as covalently linked escorting moieties, but a much wider diversity of iron binding motifs exists. This observation, coupled to the relative lack of specificity of siderophore receptors, prompted us to initiate a program to identify novel, noncatechol siderophoric structures. We screened over 300 compounds for their ability to (1) support growth in low iron medium of a Pseudomonas aeruginosa siderophore biosynthesis deletion mutant, or (2) compete with a bactericidal siderophore-antibiotic conjugate for siderophore receptor access. From these assays we identified a set of small molecules that fulfilled one or both of these criteria. We then synthesized these compounds with functional groups suitable for attachment to both monobactam and cephalosporin core structures. Siderophore-beta-lactam conjugates then were tested against a panel of Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus strains. Although several of the resultant chimeric compounds had antimicrobial activity approaching that of ceftazidime, and most compounds demonstrated very potent activity against their cellular targets, only a single compound was obtained that had enhanced, siderophore-mediated antibacterial activity. Results with tonB mutants frequently showed increased rather than decreased susceptibilities. suggesting that multiple factors influenced the intracellular concentration of the drugs.