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.

Telithromycin is active against Mycobacterium avium in mice despite lacking significant activity in standard in vitro and macrophage assays and is associated with low frequency of resistance during treatment.

The activity of telithromycin, a new ketolide, was evaluated in vitro and in vivo against Mycobacterium avium complex (MAC) strains. The MIC of telithromycin for several M. avium isolates obtained from the blood of AIDS patients ranged from 16 to >128 microg/ml (MIC at which 90% of isolates are inhibited, >128 microg/ml), and the compound did show activity in the macrophage system at concentrations greater than 8 or 16 microg/ml, but this was dependent on the MAC strain used. Telithromycin was then administered to mice infected with MAC strain 101 for 4 weeks at doses of 100, 200, or 400 mg/kg of body weight/day. Treatment with 100 and 200 mg/kg/day was bacteriostatic, but at 400 mg/kg/day telithromycin was bactericidal for MAC strains. The frequency of the emergence of resistance to telithromycin was low despite prolonged usage (12 weeks). This study demonstrates that telithromycin is active in vivo against MAC and warrants further evaluation.

Activity of moxifloxacin by itself and in combination with ethambutol, rifabutin, and azithromycin in vitro and in vivo against Mycobacterium avium.

Moxifloxacin activity against Mycobacterium avium complex (MAC) was evaluated in vitro against 25 strains. The MIC was determined to range from 0.125 to 2.0 microg/ml. In addition, U937 macrophage monolayers infected with MAC strain 101 (serovar 1) were treated with moxifloxacin (0.25 to 8 microg/ml) daily, and the number of intracellular bacteria was quantitated after 4 days. Moxifloxacin showed inhibitory activity at 0.5 microg/ml and higher. To assess the activity of moxifloxacin containing regimens in vivo, we infected C57BL bg(+)/bg(+) mice with 3 x 10(7) MAC strain 101 bacteria intravenously. One week later treatment was begun with the following: (i) moxifloxacin (50 mg/kg/day or 100 mg/kg/day), ethambutol (100 mg/kg/day), or a combination of moxifloxacin and ethambutol; or (ii) moxifloxacin (100 mg/kg/day), azithromycin (200 mg/kg/day), or rifabutin (40 mg/kg/day) as oral monotherapy; or (iii) all permutations of two-drug therapy or all three drugs in combination. All groups contained at least 14 animals, and the control group received the drug vehicle. After 4 weeks, quantitative blood cultures were obtained and the number of bacteria in liver and spleen was quantitated. Moxifloxacin, ethambutol, and azithromycin were active as single agents in liver, spleen, and blood. Rifabutin showed inhibitory activity only in the blood. Two-drug combinations containing azithromycin were no more active than azithromycin alone. Similarly, the three-drug combination was not more active than azithromycin alone in the spleen. Rifabutin did not add to the activity of any other single agent or two-drug combination. Moxifloxacin at both concentrations in combination with ethambutol was significantly more active than each drug alone.

Clarithromycin-resistant mycobacterium avium is still susceptible to treatment with clarithromycin and is virulent in mice.

Resistance to clarithromycin in breakthrough Mycobacterium avium complex (MAC) isolates typically occurs 3 to 4 months after the initiation of monotherapy in bacteremic AIDS patients. It has been suggested that continuation of clarithromycin therapy still results in clinical and microbiological improvement. To study this paradox, C57BL/6 beige mice were infected with a clarithromycin-resistant (MIC, > or =128 microg/ml) strain of MAC 101 (CLA-R MAC 101) and treated with 200 mg of clarithromycin per kg of body weight/day alone or in combination with ethambutol (100 mg/kg/day) for 2 weeks. Mice infected with a clarithromycin-susceptible strain of MAC 101 had bacterial loads reduced by 90% in the liver and 91% in the spleen (P<0.05, compared with the control). Clarithromycin treatment of CLA-R MAC 101 resulted in a 65% reduction of bacterial loads in the liver (P = 0.009) and a 71% reduction in the spleen (P = 0.009), compared with the results for the untreated control. CLA-R MAC 101 and MAC 101 (isogenic strains) had comparable growth rates in murine tissue, ruling out a loss of virulence of CLA-R MAC 101. Strains of MAC currently defined as macrolide resistant may still respond to treatment with an agent such as clarithromycin within infected tissues.

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.

Mefloquine is active in vitro and in vivo against Mycobacterium avium complex.

Despite the development of several agents, new classes of antimicrobials with activity against the Mycobacterium avium complex (MAC) are needed. Based on a broad screening of compounds, we found that mefloquine has MICs of 8 to 16 microg/ml by the BACTEC system and 16 microg/ml by broth microdilution for five MAC strains tested. An expansion of the screening with broth microdilution to 24 macrolide-susceptible strains and 6 macrolide-resistant strains determined that the MIC for all strains was 16 microg/ml. To determine the intracellular activity of mefloquine, U937 macrophage monolayers infected with MAC strain 101, 100, or 109 (serovars 1, 8, and 4) were treated with mefloquine daily, and the number of intracellular bacteria was quantitated after 4 days. Significant growth inhibition against the three MAC strains at concentrations greater than or equal to 10 microg/ml (P < 0.05) was obtained. Due to the encouraging anti-MAC activity, in vivo efficacy in beige mice infected with MAC 101 was evaluated. Animals were treated with 5, 10, 20, or 40 mg/kg of body weight daily, three times a week, twice a week, or once a week for 4 weeks, and bacteria were quantitated in blood, liver, and spleen. No toxicity was observed with any of the treatment regimens. Mefloquine had borderline bactericidal activity at a dosage of 40 mg/kg daily (100% inhibition compared with a 1-week control), and significant inhibition was obtained at dosages of 40 mg/kg three times a week, as well as 20 mg/kg daily. Mefloquine had no significant effect on bacteremia. A combination of mefloquine and ethambutol showed significantly more activity than did either drug alone in liver, spleen, and blood; the combination was also bactericidal against M. avium. Although safety is a potential concern, mefloquine and related compounds deserve further investigation as anti-MAC therapies.

Correlation of quantitative bone marrow and blood cultures in AIDS patients with disseminated Mycobacterium avium complex infection.

The relationship between Mycobacterium avium complex (MAC) infection of blood and bone marrow was studied in human immunodeficiency virus-infected patients before and during treatment. Quantitative cultures were obtained at baseline from 17 persons with newly detected MAC bacteremia. Serial blood cultures were obtained, and a second bone marrow sample was obtained at 4 or 8 weeks. At baseline, the median MAC load in bone marrow core samples was 3 log10 higher than in blood. Bone marrow MAC loads ranged widely (866-847,315 cfu/g), and no significant correlation was found between MAC load in blood and that in bone marrow core samples. MAC loads in bilateral bone marrow biopsy samples from 7 subjects were highly correlated. MAC loads declined in blood and bone marrow at similar rates during therapy, but blood was sterilized before bone marrow. Length of survival was inversely associated with initial bone marrow core MAC load but not with blood MAC load. Initiation of treatment when tissue MAC load is low may increase the likelihood of favorable clinical outcome.

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.

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.

Detection of rifampin resistance in Mycobacterium tuberculosis by use of a rapid, simple, and specific RNA/RNA mismatch assay.

An adaption of an RNA/RNA duplex, base pair-mismatch assay is capable of detecting rifampin resistance in Mycobacterium tuberculosis. The specificity and sensitivity of the mismatch assay in detecting rifampin resistance were 100% and 96%, respectively, when tested against 46 rifampin-resistant and rifampin-susceptible strains of M. tuberculosis. By use of a range of mycobacterial and nonmycobacterial prokaryote pathogens, the mismatch assay was shown to be specific for M. tuberculosis and Mycobacterium bovis. The assay is cost-effective compared with DNA sequencing and other molecular methods and is simple to perform and interpret. Furthermore, the assay can return a result within 24 h after receipt of an isolated organism and potentially can be used directly with smear-positive specimens.

Effect of ethambutol on emergence of clarithromycin-resistant Mycobacterium avium complex in the beige mouse model.

An animal model was developed for studying macrolide-resistant Mycobacterium avium complex (MAC) and to measure the effect of ethambutol on resistance. MAC-infected beige mice were given clarithromycin daily; the frequency of clarithromycin-resistant MAC after 8 and 12 weeks was 10(-3) and 10(-2), respectively. Combined ethambutol plus clarithromycin did not increase anti-MAC activity, but clarithromycin-resistant MAC was less frequent (P < .05). The frequency of clarithromycin-resistant MAC in mice receiving the combination was significantly higher than that in untreated mice. These results are consistent with two human trials, which showed that adding ethambutol reduced the frequency of clarithromycin-resistant MAC. Results of the present study suggest that with an initially high level of MAC infection, the addition of ethambutol may only delay resistance. This mouse test system will be useful for investigating the influence of the level of MAC infection and the effect of other drugs on the frequency of resistant MAC.

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).

Activities of bay Y 3118, levofloxacin, and ofloxacin alone or in combination with ethambutol against Mycobacterium avium complex in vitro, in human macrophages, and in beige mice.

Levofloxacin, ofloxacin, and Bay Y 3118 are new fluoroquinolones with variable in vitro bacteriostatic and bactericidal activities against the Mycobacterium avium complex (MAC). The potential therapeutic activities of these agents both alone and combined with ethambutol were evaluated in a human macrophage test system and in the beige mouse animal test system with MAC strain 101. Bay Y 3118 at a human-equivalent dose of 30 mg/kg/day for 4 weeks caused a significant reduction in mortality compared with that in untreated controls (P = 0.02). Bay Y 3118 also caused significant reductions in the number of MAC organisms in the blood, liver tissue, and spleen tissue compared with those in untreated controls. Levofloxacin at a human-equivalent dose of 200 mg/kg/day was associated with a significant reduction in mortality (10 versus 39%); however, treatment with either levofloxacin or ofloxacin (200 mg/kg/day) did not result in significant reductions in the numbers of MAC organisms in blood, liver, and spleen compared with those in untreated controls. When Bay Y 3118 was combined with ethambutol, there was no enhancement in therapeutic activity except in the spleen in terms of CFU per gram (reductions of 89% compared with the untreated control, 63% compared with Bay Y 3118 alone, and 72.5% compared with ethambutol alone). Levofloxacin in combination with ethambutol was more active than either drug alone in the reduction of organisms in blood, liver, and spleen. Bay Y 3118 was the most active fluoroquinolone for monotherapy of MAC infection in beige mice, and the combination of ethambutol plus either levofloxacin or ofloxacin was at least additive. In summary, this study demonstrates that quinolones, although active, are inhibitory against MAC in vivo and that there is little correlation between the activity of quinolones in vitro and the activity in mice.

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.

Internal controls as performance monitors and quantitative standards in the detection by polymerase chain reaction of herpes simplex virus and cytomegalovirus in clinical specimens.

Internal controls (IC) were produced and characterized for an HSV and a CMV PCR assay which serve both as test performance monitors and as quantitative standards. In each of the PCR assays the IC and native targets were amplified with equal efficiency and were detected with the same sensitivity, i.e. < 10 target copies. An algorithm was developed for the use of IC as a quantitative standard which entailed coamplifying a test specimen with four two-fold dilutions of the respective IC target (63-500 copies), followed by regression analysis of the relative yield of amplification products. This approach allowed the determination of both the initial virus genome copy number and the variability of the results, which provided a confidence index for the PCR assay. The relative yields of PCR products were determined by Southern blot and probe hybridization and by densitometry of digitized ethidium bromide-stained gels. Both methods produced estimations of the initial target copy numbers within +/- 40% of the expected value. Such a comprehensive analysis of an internal control for a PCR assay provides a rigorous control of test performance and permits reliable quantitative interpretation of a PCR assay result.

Efficacy of azithromycin and rifabutin in preventing infection by Mycobacterium avium complex in beige mice.

We investigated the potential of the azalide, azithromycin, and rifabutin in preventing disseminated infection due to Mycobacterium avium complex (MAC) in beige mice. Azithromycin 200 mg/kg, rifabutin (30 mg/kg or 60 mg/kg) were administered by gavage 6 days before mice were challenged orally with 10(8) cfu MAC and daily for 10 days thereafter during which time the mice were again challenged with the same inoculum on alternate days (days +1, +3, +5, +7, and +9). Sixty-four days later, the presence of bacteria in the blood and the number of viable bacteria in liver, spleen and appendix were estimated. Treatment with azithromycin and 60 mg/kg/day rifabutin but not 30 mg/kg/day, significantly decreased the incidence of bacteraemia and the number of bacteria in the appendix. The administration of azithromycin resulted in significantly fewer MAC in the liver and spleen but not in the appendix whereas the converse was true of 60 mg/kg rifabutin. Our results indicate that both azithromycin and rifabutin can prevent MAC disseminated infection, but that the azalide is more effective than the rifamycin in reducing the burden of infection.

Activity of ampicillin/sulbactam, ticarcillin/clavulanate, clarithromycin, and eleven other antimicrobial agents against anaerobic bacteria isolated from infections in children.

The activity of 14 antimicrobial agents against 253 clinical isolates of anaerobic bacteria from pediatric infections was assessed by the agar dilution method. Fifty-eight percent of the isolates were from intraabdominal sites. The drugs tested were ampicillin/sulbactam, ticarcillin/clavulanate, ampicillin, sulbactam, piperacillin, cefoxitin, cefotaxime, cefoperazone/sulbactam, clarithromycin, azithromycin, erythromycin, clindamycin, metronidazole, and chloramphenicol. Ticarcillin/clavulanate was active against all isolates. Clarithromycin was the most active macrolide; combination of this agent with its 14-hydroxy metabolite did not result in synergy. Sixty-two percent of Bacteroides fragilis group isolates, 13% of B. fragilis isolates, and 22% of peptostreptococcal isolates were resistant to clindamycin at a concentration of 4 micrograms/mL. The distribution of these strains in clinical specimens and the patterns of antimicrobial susceptibility documented were different from the findings for isolates from adults in the Los Angeles area.

In-vitro activity of oxazolidinones against Mycobacterium avium complex.

Options for treating disseminated Mycobacterium avium complex (MAC) disease have improved. However, efficacy is not always certain, resistance is common and rapidly bactericidal agents would improve efficacy and prevent resistance. Certain oxazolidinones were tested against MAC strains and inhibited growth at expected serum concentrations or lower. Activity correlated with hydrophobicity and one agent was bactericidal at concentrations two to five times greater than the MIC.

Potential role of roxithromycin against the Mycobacterium avium complex.

Until the recent experience with azithromycin and clarithromycin, macrolides were not considered to be important agents against mycobacteria. Clinical evidence is now growing that the newer 14 and 15 membered macrolide compounds have therapeutic activity against Mycobacterium avium, Mycobacterium chelonae and Mycobacterium leprae. Several years ago, when evaluating the activity of roxithromycin using one of the more virulent M. avium in our collection, the authors found that roxithromycin exerted a bacteriostatic effect in cultured human macrophages. However, in combination with tumour necrosis factor, which induces macrophage activation, roxithromycin caused enhanced intracellular killing. The significance of this finding is that tumour necrosis factor can be elaborated by activated macrophages during the course of infection. The roxithromycin doses that were chosen for these studies were less than achievable blood levels. More recently, the in vitro effect of roxithromycin against a panel of isolates from AIDS patients has been assessed and it was found that some (but not all) of the inhibitory concentrations, by the T-100 method of Inderlied, are within achievable serum levels. This, however, may not be the basis for anticipating in vivo activity since macrolide compounds are known to be concentrated within cells and particularly within phagolysosomes. Demonstration of effect in an in vitro test system is encouraging, but should be considered only as a preliminary step to careful assessments in experimental animals, such as the beige mouse, and studies in humans.