David M. Yajko

Bacteriology of the marine environment: Implications for clinical therapy

Ocean water and tissue samples were obtained from a variety of sources with phylogenetic and geographic diversity. Purified bacterial colonies were isolated and identification procedures were performed. A total of 67 isolates were recovered. Thirty-eight isolates belonged to the genus Vibrio and included six species. Twenty-four non-fermentative bacteria and four Gram-positive isolates were recovered. Antibiotic susceptibility testing showed that while the non-fermentative marine bacteria generally were susceptible to the antibiotics tested, marine Vibrio species were relatively resistant to a wide variety of antimicrobials. Antibiotics effective against all species included imipenem, trimethoprim/sulfamethoxazole, and chloramphenicol. Further recommendations for treatment are based on sensitivity in culture. Some isolates failed to grow in the medium used for susceptibility testing. Because commercial test kits may not yield accurate identifications of bacteria, the acquisition of antimicrobial susceptibility data gains added importance.

Therapeutic implications of inhibition versus killing of Mycobacterium avium complex by antimicrobial agents.

Patients with the acquired immune deficiency syndrome (AIDS) with disseminated Mycobacterium avium infection have responded poorly to treatment with rifabutine (Ansamycin) and clofazimine, in spite of the good in vitro response of M. avium to these antimicrobial agents. We compared the ability of these and other antimicrobial agents to kill versus the ability to inhibit the growth of strains of the M. avium complex isolated from patients with AIDS. Killing curve experiments showed that the concentrations of rifabutine and clofazimine needed to kill two log units of M. avium are at least 32 times greater than the concentrations needed to inhibit growth. Little or no killing occurred at concentrations of these antimicrobial agents that are achievable in serum. In contrast, five of seven strains tested were killed by ciprofloxacin at concentrations that can be achieved in serum. Ciprofloxacin should be studied further for possible use in the treatment of M. avium infections.

Antimicrobial synergism against Mycobacterium avium complex strains isolated from patients with acquired immune deficiency syndrome.

Pairs of 11 antimicrobial agents were tested in vitro for their ability to act synergistically against three strains of Mycobacterium avium complex isolated from patients with acquired immune deficiency syndrome. From the combinations tested, four drugs (ethambutol, rifampin, ciprofloxacin, and erythromycin) were selected for more extensive study against 20 strains of M. avium complex. The inhibitory and killing synergism obtained with combinations of two, three, or four drugs was assessed by determining the fractional inhibitory concentration index and fractional bactericidal concentration index. Inhibitory synergism occurred against 90 to 100% of the strains for all drug combinations in which ethambutol was included. Killing synergism occurred against 85 to 95% of the strains when ethambutol was used in combinations which included either rifampin or ciprofloxacin. However, killing synergism occurred against only 45% of the strains when drugs were tested at concentrations that can be obtained in patient serum. In other experiments, rifabutin (Ansamycin) gave results that were comparable to those obtained with rifampin. Clofazimine did not show synergistic killing activity at a concentration that is achievable in serum for any of the drugs tested. Our results indicate that there is considerable variability in the antimicrobial susceptibility of M. avium isolates obtained from patients with acquired immune deficiency syndrome. This variability could have significant impact on the clinical response to various therapies.

Broth microdilution testing of susceptibilities to 30 antimicrobial agents of Mycobacterium avium strains from patients with acquired immune deficiency syndrome.

A total of 31 strains of Mycobacterium avium complex isolated from patients with acquired immune deficiency syndrome were tested for susceptibility to 30 antimicrobial agents by using microdilution trays containing dried antimicrobial agents. MICs were determined over a period of 7 days of growth in a broth medium (7HSF) that is equivalent to 7H11 agar. MICs obtained by this method showed good agreement with MICs determined by the agar dilution method. Strains could be divided into two groups by their antimicrobial susceptibility patterns. All group 1 strains (8 of the 31 strains tested) were at least moderately susceptible to inhibition by a variety of beta-lactam antimicrobial agents, including amoxicillin-clavulanic acid and cefmenoxime. Group 2 strains (23 of 31) were susceptible only to amikacin (22 of 23 strains). All 31 strains were resistant to oxacillin, clindamycin, erythromycin, tetracycline, chloramphenicol, vancomycin, nitrofurantoin, and aztreonam at the highest concentration of antimicrobial agent present in the microdilution trays. The addition of Tween 80 to 7HSF broth increased the susceptibility of M. avium complex to many of the antimicrobial agents tested. Killing of M. avium complex (i.e., less than or equal to 1% survival after 7 days) was found to vary for different strains and antimicrobial agents. Killing of some strains by amoxicillin-clavulanic acid, carbenicillin, azlocillin, cefmenoxime, cefotaxime, amikacin, and ampicillin occurred at concentrations of antimicrobial agent that are achievable in serum. Further studies are needed to determine whether any of these antimicrobial agents has activity against M. avium complex cells that have been ingested by macrophages.

Characterization of Strains of Viridans Streptococci by Deoxyribonucleic Acid Hybridization and Physiological Tests

Relationships among selected species of viridans streptococci were studied by using deoxyribonucleic acid (DNA)-DNA hybridization and biochemical and serological tests. The results of hybridization experiments performed with DNA immobilized on membrane filters and index DNA made radioactive with [methyl-3H]thymidine indicated that Streptococcus intermedius ATCC 27335T (type strain), Streptococcus constellatus ATCC 27823T, Streptococcus mitis ATCC 9895, “Streptococcus MG-intermedius” CDC SS 899, group F strain MGH 8153, and group F strain SFGH 879 are closely related to each other genetically, even though physiological tests indicated that this is a relatively heterogeneous group of organisms. The relative binding ratios for these six strains ranged from 60 to 99%, and the majority of values were greater than 85%, indicating that these strains should be considered members of the same species. Type strain ATCC 33399 (called S. mitis) is not a typical S. mitis strain based on biochemical test results, and only moderate relatedness (41%) was observed in hybridization experiments performed with a typical S. mitis strain (strain SS 429). Streptococcus sanguis I ATCC 10556T and S. sanguis II ATCC 10557 do not belong in the same species. A low relative binding ratio (13%) for these strains confirmed similar findings by previous investigators.

In vitro susceptibility of Mycobacterium avium complex to the new fluoroquinolone sparfloxacin (CI-978; AT-4140) and comparison with ciprofloxacin.

We tested the activity of the new fluoroquinolone sparfloxacin (CI-978; AT 4140) against 30 strains of Mycobacterium avium complex (MAC) isolated from patients with acquired immune deficiency syndrome. MICs of sparfloxacin (range, less than or equal to 0.06 to 4 micrograms/ml) were lower than MICs of ciprofloxacin for all 30 strains, and MBCs for acid-fast bacteria were lower for 28 of the 30 strains. In synergism experiments using 10 strains of MAC, fractional inhibitory concentration indices revealed that the combination of sparfloxacin plus ethambutol was synergistic against 9 strains, and the three-drug combination of sparfloxacin plus ethambutol plus rifampin was synergistic against all strains. In the absence of ethambutol, the combination of sparfloxacin plus rifampin appeared to be antagonistic against three of the MAC strains.

Comparison of the intracellular activities of clarithromycin and erythromycin against Mycobacterium avium complex strains in J774 cells and in alveolar macrophages from human immunodeficiency virus type 1-infected individuals.

The intracellular activities of clarithromycin and erythromycin, alone and in combination with other antimicrobial agents, were tested against Mycobacterium avium complex (MAC) strains inside mouse J774 cells and inside alveolar macrophages obtained from human immunodeficiency type 1-infected individuals. Clarithromycin alone had greater intracellular activity than erythromycin alone, and drug combinations that included clarithromycin were usually more active than combinations that included erythromycin.

Genetic Similarity among Mycobacterium avium Isolates from Blood, Stool, and Sputum of Persons with AIDS

Large-restriction-fragment pattern comparison of Mycobacterium avium from 85 blood, stool, and respiratory specimens from 25 human immunodeficiency virus-infected San Francisco patients revealed 4 strains that infected multiple people (3 groups of 2 patients and 1 group of 3 patients). Most patients harbored a single M. avium strain, but 2 strains were recovered from 8 patients. The significance of recovering 2 strains is not clear, since the second strain was seldom recovered more than once. The strain recovered from blood was recovered from stool of 4 patients and respiratory secretions of 6 patients >4 weeks before detection of bacteremia, indicating that the intestinal and respiratory tracts are entry portals from which M. avium can disseminate. M. avium from 21 cities outside of California served as controls. Thus, a single M. avium strain can cause disseminated infection in multiple patients. This may represent infection from a common environmental source or person-to-person spread.

Effects of antimicrobial agents on survival of Mycobacterium avium complex inside alveolar macrophages obtained from patients with human immunodeficiency virus infection.

Measurements of the activities of antimicrobial agents against the Mycobacterium avium complex (MAC) usually do not take into consideration the intracellular location of the organism. A recent study using mouse macrophage continuous cell line J774 (D. M. Yajko, P.S. Nassos, C. A. Sanders, and W. K. Hadley, Am. Rev. Respir. Dis. 140: 1198-1203, 1989) showed that certain combinations of antimicrobial agents are able to kill MAC inside macrophages and suggested that the J774 cell line could be used as a model for screening of drugs for intracellular activity against MAC. As a test of the validity of this model, alveolar macrophages were isolated from the bronchoalveolar lavages of 36 patients who had AIDS or an AIDS-related condition or were considered to be at risk for AIDS. The macrophages were infected with MAC and then treated with a drug or drug combination for 48 to 72 h. Survival of MAC was measured over time in drug-treated macrophages and untreated control macrophages. No single drug or two-drug combination that was tested was able to cause a decrease in the survival of every one of the MAC strains used in the study. However, several three-drug combinations that had been shown to cause a decrease in survival of all MAC strains inside J774 cells also caused a decrease in survival of all MAC strains inside alveolar macrophages from patients. The good agreement between these results and those obtained previously with J774 cells gives further evidence of the usefulness of the simpler J774 model for screening of drugs for intracellular activity against MAC.