Molecular characterisation of Haemophilus influenzae type a and untypeable strains isolated simultaneously from cerebrospinal fluid and blood: novel use of quantitative real-time PCR based on the cap copy number to determine virulence.

This study investigated the genetic structure of the cap region of an isolate of Haemophilus influenzae serotype a (Hia) from the cerebrospinal fluid (CSF) of a child with meningitis. In addition, the genetic structure of the cap region of a non-serotypeable H. influenzae isolate, obtained simultaneously from the blood of the same patient, was determined. According to restriction fragment length polymorphism analysis, the CSF and blood isolates were identical, with the exception of a single band shift of c. 35 kb. PCR analyses suggested that the CSF isolate possessed the IS1016-bexA gene and cap region II, whereas the blood isolate only had the IS1016 element. Furthermore, Southern analysis of DNA from both isolates showed that the CSF isolate carried the cap gene(s), while the blood isolate did not. Using a novel quantitative real-time PCR approach for determining the cap copy number, it was demonstrated that the CSF isolate had two intact tandem repeats of the cap gene containing three copies of IS1016, whereas the blood isolate had only one copy of IS1016. This study provided evidence that H. influenzae serotypes other than serotype b can cause serious disease, and that the virulence of these non-serotype b strains relates primarily to the cap gene copy number and the structure of the cap locus. Therefore, the quantitative real-time PCR assay described in this study should be useful for the rapid and definitive identification of strains of H. influenzae type a that represent a risk for serious disease.

Activity of ciprofloxacin and other fluorinated quinolones against mycobacteria.

The new fluorinated quinolones display interesting but variable activity against mycobacteria. Almost all compounds tested (ciprofloxacin, ofloxacin, enoxacin, norfloxacin, difloxacin, CI-934, A-56620, and megalone) inhibit Mycobacterium tuberculosis at achievable serum concentrations, with ciprofloxacin and ofloxacin most active by weight (minimal inhibitory concentration at which growth of 90 percent of strains is inhibited is equal to 1 microgram/ml or less). The growth of Mycobacterium kansasii, Mycobacterium xenopi, and Mycobacterium fortuitum is also well inhibited by these agents in the same range of concentrations. Activity against the Mycobacterium avium complex is method-dependent, with growth of perhaps one third of the strains isolated from patients with the acquired immune deficiency syndrome inhibited by ciprofloxacin. Determination of individual drug efficacy data in experimental mycobacterial infections is not a practical goal. However, combination therapy studies are in progress using murine models of both M. tuberculosis and M. avium challenges. Ofloxacin has been used with some success in human patients with pulmonary tuberculosis. Oral administration may be an important advantage, and, when used in combination with other active agents, the new quinolones may have a useful role in treating mycobacterial infections.

Imipenem/cilastatin treatment of bacterial meningitis in children.

The safety and efficacy of imipenem/cilastatin were evaluated in 21 children, ages 3 to 48 months, with bacterial meningitis. Eradication of bacteria from the cerebrospinal fluid was demonstrated within 24 hours of antibiotic therapy in all but 2 patients who had Haemophilus influenzae type b meningitis and ultimately achieved bacteriologic cure after 2 to 3 days of imipenem/cilastatin therapy. Cerebrospinal fluid penetrations of imipenem and cilastatin were determined at various times after drug administration with mean cerebrospinal fluid: serum ratios of 14 and 10% for imipenem and cilastatin, respectively. The study was terminated when 7 (33%) patients developed seizure activity after antibiotic therapy was administered. The usefulness of imipenem/cilastatin for the treatment of bacterial meningitis in children may be limited by a possible increased incidence of drug-related seizure activity.

Effect of Mycobacterium avium infection on the influx, accumulation, and efflux of KRM-1648 by human macrophages.

KRM-1648 is a new benzoxazinorifamycin with activity in vitro and in vivo against organisms of the Mycobacterium avium complex. We investigated the ability of 14C-KRM-1648 to concentrate within human monocyte-derived macrophages in vitro. KRM-1648 is rapidly taken up by uninfected macrophages, with 90% of the initial concentration added to the monolayer found within macrophages by 1 h and approximately 80% at 2 h. Comparable results were obtained in assays using macrophages that have been infected with an AIDS-related strain of M. avium for 24 h. In contrast, macrophages infected with M. avium for 3 days, showed an impaired ability to concentrate KRM-1648, primarily because of a significant efflux of the antibiotic (intracellular concentration of 86% of the available drug was present within macrophages at 1 h vs. 47% at 2 h). Daily administrations of KRM-1648 to a macrophage monolayer for 3 consecutive days resulted in significant accumulation of the drug within phagocytic cells. Although the efflux was greater in M. avium-infected macrophages than in uninfected cells, consecutive administration of KRM-1648 led to a total intracellular accumulation of drug that exceeded the initial level and appeared to continue to accumulate. The ability of KRM-1648 to rapidly accumulate in human macrophages, including M. avium-infected cells, may explain, in part, the improved therapeutic effectiveness in animal models against M. avium and M. tuberculosis.

Diagnostic efficacy of a nasotracheal protected specimen brush in patients with suspected bacterial pneumonia.

The diagnostic yield and safety of a novel nasotracheal protected specimen brush (PSB) were evaluated in 15 nonintubated adult patients with suspected bacterial pneumonia. A double-catheter PSB was passed directly through the anesthetized nose and into the trachea without bronchoscopy or fluoroscopy. Endotracheal brushing was performed in less than 10 sec, and the brush was immediately processed for Gram staining and quantitative aerobic and anaerobic cultures. According to clinical follow-up and response to therapy, 11 episodes of bacterial pneumonia and five cases of nonbacterial lung disease were established. The PSB Gram stain confirmed lower respiratory sampling in all cases. The PSB cultures indicated respiratory pathogens in 9/11 (82%) cases of pneumonia, with greater than 10(3) colony-forming units (cfu)/ml in all but two specimens. All patients with pneumonia responded to specific antibiotics. All patients with nonbacterial disorders had PSB cultures of less than 10(3) CFU/ml, and their pulmonary processes improved without antibiotic therapy. The procedure was well tolerated, although two patients had transient bronchospasm or apnea. Experience with the nasotracheal PSB is limited, but the procedure appears to be a reliable and relatively safe alternative diagnostic method in selected patients with suspected bacterial pneumonia. Quantitative cultures are necessary to improve its diagnostic accuracy.

The role of copper on ethambutol's antimicrobial action and implications for ethambutol-induced optic neuropathy.

The principal side effect of the antimycobacterial agent ethambutol (EMB) is an optic neuropathy with clinical features very similar to a mitochondrial hereditary optic neuropathy (Leber's). The mechanism of EMB-induced optic neuropathy may be EMB's chelation of copper, thereby precluding normal cytochrome c oxidase activity and mitochondrial metabolism in the optic nerve. Before attempting to use therapeutic copper to replenish endogenous stores in an attempt to preclude EMB-induced optic neuropathy, we wished to determine whether EMB is still effective against mycobacteria in the presence of copper. EMB and copper, alone and in combination, were tested against six strains of Mycobacterium tuberculosis and five strains of Mycobacterium avium using a radiometric broth macrodilution assay. Copper did not effect EMB's antimicrobial actions against either species of mycobacteria. This in vitro study suggests that if copper were given to patients to prevent EMB-induced optic neuropathy, it would not compromise EMB's bacteriostatic properties.

In vitro activity of lomefloxacin as compared with ciprofloxacin.

Lomefloxacin (SC47111, Searle) is a difluorinated quinolone with a comparatively long half-life and high serum concentration. This agent has good in vitro activity against Enterobacteriaceae (MIC90 = less than 2 micrograms/ml) and staphylococci (MIC90 = less than 2 micrograms/ml); the activity against Pseudomonas spp. and Pseudomonas aeruginosa is moderate to poor. On a weight basis, lomefloxacin is less active than ciprofloxacin; however, based on the ratio of the serum concentration to MIC, the activity of lomefloxacin is nearly equivalent to ciprofloxacin against many bacteria, with the exception that ciprofloxacin has good activity against most pseudomonads. Also, lomefloxacin was active against a variety of bacteria that were resistant to aminoglycosides and/or third-generation cephalosporins. A majority of strains of the Mycobacterium avium complex, isolated from AIDS patients with disseminated disease, were found to be resistant to lomefloxacin (MIC90 = greater than 8 micrograms/ml).

Antimicrobial susceptibility of clinical isolates of Haemophilus influenzae to ampicillin-sulbactam.

A total of 1092 clinical isolates of Haemophilus influenzae (306 type b; 786 non-type-b), from five medical centers were obtained during 1987 and 1988. Disk diffusion antimicrobial susceptibilities were obtained for all isolates, and broth microdilution susceptibilities were obtained for 502 isolates. Beta-lactamase was produced by 34.3% of type-b and 22.1% of non-type-b isolates, with some geographic variations. Using disk diffusion antimicrobial susceptibility testing, all isolates were susceptible to ampicillin-sulbactam, ceftriaxone, cefuroxime, and rifampin; two isolates were resistant to chloramphenicol. Whether tested using a fixed ratio of ampicillin to sulbactam of 2:1 or a fixed concentration of sulbactam, the ampicillin-sulbactam combination demonstrated good activity against clinical isolates of H. influenzae. Only 8 of the 1092 isolates did not produce beta-lactamase but demonstrated MICs of greater than or equal to 2 micrograms/ml for ampicillin.

Isolation of two subpopulations of Mycobacterium avium within human macrophages.

Mycobacterium avium is an intracellular pathogen that is associated with disseminated infection in acquired immunodeficiency syndrome (AIDS) patients. Human monocyte-derived macrophages were infected with M. avium strain 101 and a quinolone (Bay y 3118) was used at 8 micrograms ml-1, a concentration that kills growing bacteria but fails to eliminate static organisms. Infected monolayers were treated with Bay y 3118 for 4 days and viable bacteria obtained from the lysis of macrophages were used to infect other macrophages without passage in media. The procedure was repeated five times, after which seven different subpopulations that failed to grow within macrophages were identified. While the DNA fingerprinting confirmed that all came from the same strain, three protein profiles were observed. Static subpopulations were not killed by cytokine-stimulated macrophages, in contrast to the replicating subpopulation. Three of the static subpopulation strains were shown to be auxotrophic for glutamic acid or methionine. All seven non-duplicating subpopulation strains grew well in complete 7H10 agar. The importance of a static subpopulation of M. avium within macrophages is presently unknown. It is possible, however, that the non-growing bacteria would persist within macrophages.

Synergistic antiviral effect of PEG-asparaginase (ONCASPAR), with protease inhibitor alone and in combination with RT inhibitors against HIV-1 infected T-cells: a model of HIV-1-induced T-cell lymphoma.

We evaluated the anti-HIV-1 activity of the T-cell-specific protein inhibitor PEG-asparaginase (PEG-ASNase) in human HIV-1-infected T-cells. We further examined the drug synergism between PEG-ASNase and the protease inhibitor Saquinavir (SAQ), both alone and in combination with nucleoside analog reverse transcriptase inhibitors (NRTI). Our drug synergism studies served as a model for an HIV-induced T-cell lymphoma. Phytohemagglutinin [PHA(+)] stimulated T-cells were infected with HIV-1 and then treated with one or more drugs 90 minutes from the viral exposure. To measure inhibition of viral replication, we examined HIV-1 RT and HIV-1 RNA in the supernatant and intracellularly on day 7 post-infection and drug treatment. Last, we examined the effect of administering drugs immediately after HIV-1 infection of T-cells to simulate treatment after an accidental exposure to the virus. PEG-ASNase, even when used alone, has anti-HIV-1 activity in PHA(+)-stimulated T-cells due to inhibition of protein synthesis. When the drug was used with SAQ, the combination was synergistic in inhibiting HIV-1 RT and RNA in the supernatant and intracellularly by 2.5 log10 in comparison with controls. PEG-ASNase and SAQ were even more effective in inhibiting HIV-1 replication when combined with the NRTI inhibitors azidothymidine (AZT) and (-)-beta-2',3'-dideoxy-3'-thiacytidine (3TC, lamivudine). The addition of ribonucleotide reductase inhibitor, 2-methyl-1H-isoindole-1,3-dione (MISID), further potentiated the antiviral effect of the regimen. HIV-1 RT and RNA analyses showed that the administration of the PEG-ASNase + SAQ drug combination immediately following exposure to HIV-1 completely inhibited the infection of T-cells in our in vitro T-cell model. From these results we conclude that PEG-ASNase is a valuable T-cell-specific protein inhibitor against HIV-1 infection, when used singly or in combination with a protease inhibitor, an RT inhibitor and an RR inhibitor. Since PEG-ASNase is a drug of choice for the treatment of T-cell lymphomas, a combination regimen containing PEG-ASNase could be very effective in the treatment of HIV-1-induced T-cell lymphoma and possibly AIDS. Future studies are needed in HIV-infected and/or HIV-induced T-cell lymphoma patients to investigate these findings.

Antimycobacterial susceptibility testing: present practices and future trends.

Along with the worldwide renewed interest in tuberculosis, the high incidence of non-tuberculous disease in HIV-infected patients and the continuing problem of disease caused by rapidly growing mycobacteria, there has been a renewed interest in mycobacterial susceptibility testing. This renewed interest stems from the needs both to provide accurate, reliable and timely susceptibility test information for the management of patients infected with these mycobacteria and to identify new and more effective antimycobacterial agents. Indeed, many new agents have already been identified and are currently used to treat these infections, but the conventional susceptibility testing methods, in many instances, have not been adequately evaluated for these new agents. Perhaps the time has come to give consideration to abandoning such time-honored practices and criteria as the proportion method, the 99% threshold, and "critical concentrations". New methods of susceptibility testing are in various stages of development and many of these methods have advantages and improvements over the existing methods. There is an increased understanding of the pathobiology of mycobacterial infections and an increased understanding of the pharmacokinetics of antimycobacterial agents and the mechanisms of action and resistance. This article gives an overview of the present practices and an assessment of the current needs and potential for the near future of antimycobacterial susceptibility testing.

Glucose metabolism and dimorphism in Mucor.

Mucor racemosus fermented glucose to ethanol, carbon dioxide, and glycerol. When this fungus was grown anaerobically in either the yeast or mycelial form, the catabolism of glucose was very similar. Yeast cells shifted to aerobic conditions maintained a high flux of glucose carbon through the glycolytic and pentose phosphate pathways. Mycelial cells grown aerobically catabolized glucose in a manner consistent with a respiratory metabolism. Although there was no consistent pattern of glucose metabolism in the mycelial form of Mucor, growth in the yeast form consistently was correlated with a high flux of glucose carbon through the catabolic pathways.

Growth of Klebsiella aerogenes on xylitol: implications for bacterial enzyme evolution.

When Klebsiella aerogenes was grown in continuous culture with xylitol. an unnatural pentitol, as the growth limiting substrate, the structural gene which codes for ribitol dehydrogenase, an enzyme which gratuitously catalyzes the oxidation of xylitol to D-xylulose, was duplicated. It appears that the duplication mechansim only duplicates the gene which is subjected to selective pressure and not any of the other closely linked genes. The degree to which the ribitol dehydrogenase gene is duplicated does not appear to be strictly correlated with the ability to grow faster on xylitol. Duplication mutants do, in fact, grow faster than their parent strain, but when challenged to grow at even higher growth rates there is a catabolic repression of enzyme activity. Thus a situation is created in which a structural gene is duplicated in response to selective pressure; these mutants can grow faster on the new substrate, but faster growth results in a "silencing" of a portion of the genes by catabolite repression.

Low prevalence of Chlamydia trachomatis in the oropharynx of adolescents.

This study determined the prevalence rates of Chlamydia trachomatis infection in the oropharynx of adolescents with and without pharyngitis. One hundred teenagers with and without symptoms of pharyngitis were cultured for C. trachomatis. Swabs were taken from the oropharynx and tonsillar areas for direct fluorescent antibody assay and cell culture. Of the 100 adolescents enrolled in the study 55% had a history of pharyngitis and 45% had no pharyngeal symptoms; 29% were male and 71% were female. The mean age was 15.5 years. Forty-two percent had a history of sexual intercourse, and 11.5% described a history of oral-genital sex. Only one 14-year-old female with a 2-week history of a sore throat had a positive culture. Her direct fluorescent antibody assay was inconclusive and the throat culture for Group A Streptococcus was negative. She had no history of sexual activity. The overall prevalence rate in the 100 adolescents was 1% with a 1.8% prevalence in the symptomatic group and 0 in the asymptomatic group. Of the symptomatic adolescents 39 had cultures for Group A Streptococcus and 8 (20.5%) were positive. The results of this study suggest that C. trachomatis is a rare inhabitant of the oropharynx in adolescents and is not a common cause of pharyngitis.

Effect of the source of Mueller-Hinton agar and resistance frequency on the detection of methicillin-resistant Staphylococcus aureus.

Inconsistencies in the results of disk diffusion tests of oxacillin against Staphylococcus aureus that occurred when using commercially prepared Mueller-Hinton agar from different sources led us to evaluate the ability of media from different sources to detect resistance to oxacillin, methicillin, and nafcillin in S. aureus. Mueller-Hinton agar from five manufacturers was prepared in our laboratory and used for standard disk diffusion and agar dilution tests. Ten oxacillin-resistant S. aureus isolates, of which three were definitive-resistant and seven were occult-resistant, were examined. All definitive-resistant strains were resistant to all three antimicrobial agents on four out of five agars. The occult-resistant strains were consistently detected as resistant on only one of the agars. With only slight differences, oxacillin, methicillin, and nafcillin resistance was more readily detected by disk diffusion and agar dilution when initially incubated at 30 degrees C, and extended incubation improved the detection. The frequency of resistance within a population of occult-resistant cells was low compared with the frequency within a population of definitively resistant cells. The heterogeneity of colony morphology and apparent growth rates within a population of occult-resistant cells contributed to the problem of detecting some resistant isolates. Definitive-resistant isolates were characterized by a very high and stable frequency of resistance. Occult-resistant strains were characterized by a lower frequency of resistance, although the true frequency of resistance may be difficult to ascertain because of heterogeneity in growth rates.

Rapid detection of mutations associated with macrolide resistance in Mycobacterium avium complex.

Macrolide resistance in Mycobacterium avium can be detected with an adaption of a commercially available RNA/RNA duplex mismatch assay (Ambion, Austin, Tex.). The sensitivity and specificity values for the assay were 100% when evaluated against 41 macrolide-resistant and -susceptible strains of M. avium. Resistant subpopulations of approximately 20% could be readily detected. The assay is simple to perform and interpret, inexpensive, and rapid (< 24-h turnaround).

Genetic basis of macrolide resistance in Mycobacterium avium isolated from patients with disseminated disease.

Clarithromycin (CLM) and azithromycin (AZM) are important agents in the treatment of disseminated Mycobacterium avium complex disease; however, monotherapy with these macrolides often leads to clinically significant resistance. The underlying resistance mechanism was investigated by comparing 23S rRNA gene sequences in the domain V region of 10 CLM-susceptible strains included in this study. The only differences in the domain V sequences associated with CLM resistance were at position 2274 of the complete M. avium 23S rRNA gene (GenBank accession no. X74494). All the CLM-susceptible strains had an A residue at this site, whereas seven of the eight CLM-resistant strains had either a C, G, or T. Four of these seven CLM-resistant strains emerged during monotherapy with CLM and two emerged during AZM monotherapy, showing that resistance selected by either macrolide was associated with mutation of the 23S rRNA gene. Thermodynamic analysis of secondary rRNA structure suggests that the observed mutations cause an alteration in free energy associated with rRNA folding, which may result in a localized conformation change in assembled ribosomes. Such a shift may be important in the resistance of ribosomes to the effects of macrolides. This study therefore establishes a link between mutations within the 23S rRNA gene and clinically significant macrolide resistance in M. avium and also identifies a possible molecular mechanism of resistance at the level of the ribosome.