Abstract
Isolates of the Mycobacterium avium complex were examined for hemolysin expression. Only invasive isolates of M. avium were observed to be hemolytic (P < 0.001), with activity the greatest for isolates of serovars 4 and 8. Thus, M. avium hemolysin appears to represent a virulence factor necessary for invasive disease.
Organisms of the Mycobacterium avium complex (MAC) are infrequent pathogens of humans. Among immunocompetent hosts, MAC primarily represents a respiratory pathogen, especially for individuals with chronic obstructive pulmonary disease, and may cause pulmonary fibrosis or cavitary lung disease (1, 5, 12). In contrast, among immunosuppressed individuals with human immunodeficiency virus (HIV) infection (2, 10) and CD4 lymphopenia (4), disseminated infection with MAC is common.
However, numerous studies have suggested a role for expressed mycobacterial products in the pathogenesis of infection. We have previously shown that M. avium expresses a magnesium-dependent, cell wall-associated hemolysin (9) and reported on the partial purification of this protein (8). Analogous to Listeria monocytogenes (11), we postulated that hemolysin expression by M. avium is necessary for intracellular survival. In this report, the epidemiology of hemolysin expression is described for invasive and noninvasive isolates of MAC.
(This study was presented in part at the 32nd Annual Meeting, Infectious Diseases Society of America, Orlando, Fla., 7 to 9 October 1994.)
Invasive isolates of MAC (n = 28) included 19 isolates of M. avium and 2 isolates of M. intracellulare cultured from individuals with AIDS (1a) and 4 isolates of M. avium and 3 isolates of MAC X cluster (3) cultured from HIV-seronegative patients (4). Respiratory isolates (n = 32) included 12 isolates of M. avium and 15 isolates of M. intracellulare cultured from elderly individuals and 3 isolates of M. avium and 2 isolates of M. intracellulare from immunosuppressed patients (4). HIV-seronegative patients met the American Thoracic Society criteria for pulmonary mycobacterial disease (1). Insufficient clinical data was available for the group of isolates cultured from elderly patients to apply these criteria; however, in many cases individuals had repeatedly positive sputa.
While false-positive samples cannot be absolutely excluded, all initial patient samples were inoculated onto solid media, which limits cross-contamination. Moreover, many patients had multiple positive cultures yielding the same strain by pulsed-field gel electrophoresis (14, 15). Pulsed-field gel electrophoresis analysis showed that each of the isolates described above represented different strains.
In prior studies, we identified clusters of patients infected with common strains of MAC (14, 15). Four additional isolates from these clusters and the environmental (potable hot water) source isolate for one cluster (15) were also analyzed.
Organisms were inoculated into 7H9 broth, incubated at 37°C to an absorbance at 600 nm (A600) of 0.2, washed once with Alsever’s solution (75 mM NaCl, 25 mM sodium citrate, 110 mM glucose, pH 6.1), resuspended in Alsever’s solution to 5 × 109 CFU/ml, and immediately assayed.
Hemolysis was determined in triplicate in 5-ml sterile, polystyrene snap-top tubes (USA Scientific, Ocala, Fla.) containing 3 ml of Alsever’s solution; 10 mM CaCl2; 10 mM MgSO4; 25 μl of washed, defibrinated sheep erythrocytes (Adams Scientific, West Warwick, R.I.); and 5 × 107 CFU of mycobacteria. Sample tubes were incubated for 6 h at 37°C and then gently mixed, and the erythrocytes were pelleted (700 × g for 10 min). The A520 of 1 ml of supernatant was determined spectrophotometrically (Spectronic 1001 spectrophotometer; Bausch and Lomb, Rochester, N.Y.) in glass cuvettes. Tubes containing either no bacteria or the prototype hemolytic strain 920A-7 were included as negative and positive controls, respectively. Hemolysis was defined as a net absorbance above 0.045: 0.045 to 0.060 was considered weakly hemolytic, 0.061 to 0.080 was considered moderately hemolytic, and >0.080 was considered strongly hemolytic.
The range of hemolysis for individual isolates typically yielded standard deviations of ≤5% (data not shown). Day-to-day variations were not greater than 10% (data not shown).
Except for some isolates (14, 15), serotyping was performed at the Laboratory of Microbiology and Pathology, Queensland Health, Brisbane, Australia.
Of the 23 isolates of M. avium causing disseminated disease, 14 (61%) were hemolytic (Table 1). The frequencies of hemolysis were similar for isolates cultured from individuals with or without AIDS. In contrast, only 1 (7%) of 15 M. avium respiratory isolates was hemolytic (P < 0.001, Fischer’s exact test). None of the 19 isolates of M. intracellulare were hemolytic (P < 0.001, Fischer’s exact test). One of three isolates of MAC X cluster was hemolytic.
TABLE 1.
Expression of hemolysin by clinical isolates of MAC
| Clinical syndrome | No. of hemolysin-expressing isolates/total no.
|
||
|---|---|---|---|
| M. avium | M. intracellulare | X cluster | |
| Disseminated infection | |||
| AIDS | 11/19 | 0/2 | |
| Non-AIDS | 3/4 | 1/3 | |
| Localized pulmonary infection: non-AIDS | 1/15 | 0/17 | |
For 34 (91%) isolates of M. avium, the serovar was determined (Table 2). Serovars 1, 4, and 8 predominated among both invasive and pulmonary strains, representing 60 and 64% of typed strains, respectively. Only isolates of serovar 4 or 8 expressed moderate or strong hemolysis, whereas other serovars expressed weak or no hemolysis (P < 0.001).
TABLE 2.
Hemolysis of human isolates of M. avium in relation to serovar
| Strain type and serovara | No. of hemolytic strains/no. tested | No. of strains with indicated level of hemolysis
|
||
|---|---|---|---|---|
| Strong | Moderate | Weak | ||
| Invasive | ||||
| 1 | 0/1 | 0 | 0 | 0 |
| 4 | 4/6 | 2 | 1 | 1 |
| 6 | 0/1 | 0 | 0 | 0 |
| 8 | 4/5 | 3 | 1 | 0 |
| 9 | 0/1 | 0 | 0 | 0 |
| 21 | 1/2 | 0 | 0 | 1 |
| Durkinb | 0/1 | 0 | 0 | 0 |
| Autoagglutinable | 1/1 | 0 | 0 | 1 |
| Nontypeable | 2/2 | 0 | 1 | 1 |
| Pulmonary | ||||
| 1 | 0/4 | 0 | 0 | 0 |
| 4 | 1/4 | 0 | 0 | 1 |
| 8 | 0/1 | 0 | 0 | 0 |
| 1-8-21 | 0/1 | 0 | 0 | 0 |
| Autoagglutinable | 0/3 | 0 | 0 | 0 |
| Nontypeable | 0/1 | 0 | 0 | 0 |
For the majority of isolates, serovars were determined by one of us (D. Dawson). Additional isolates were also typed by the Centers for Diseases Control and Prevention, Atlanta, Ga.
Serovar Durkin as defined by D. Dawson (3a).
To determine the consistency of hemolysin expression, isolates of M. avium representing the same strain from different patients were analyzed. For one pair of isolates, both isolates were strongly hemolytic, and for a second pair, both were nonhemolytic. For a third cluster of three isolates, two were strongly hemolytic and one was nonhemolytic; the matching environmental strain was also strongly hemolytic.
We found a statistically significant association between disseminated infection and hemolysin expression by M. avium. Moreover, this observation was independent of the host immune status. While hemolysin expression was not universal among invasive isolates, host factors such as gastrointestinal mucosal defects and severe immunosuppression may have allowed less virulent organisms to gain entry to the bloodstream and cause disease.
The fact that no isolate of M. intracellulare was hemolytic may represent a true difference in pathogenicity between M. avium and M. intracellulare consistent with the observation that >95% of invasive isolates of MAC represent M. avium (5, 13).
The mode of action of hemolysin on mammalian membranes is unknown; however, in other bacteria phospholipase may exhibit cytolytic activity. Genes encoding phospholipase C and phospholipase D cloned from M. tuberculosis (6) conferred a hemolytic phenotype to Escherichia coli (6). However, homologous DNA was not present in M. avium (6, 7). Whether M. avium hemolysin exhibits phospholipase activity is currently under investigation.
Thus, our data suggests that hemolysin expression by M. avium may have a role in the pathogenesis of invasive disease. The observation that hemolysin expression was greatest for isolates of serovar 4 or 8 is of interest, considering the overrepresentation of these serovars in disseminated disease, and may represent clonal segregation of this putative virulence factor.
Acknowledgments
This work was supported in part by the Medical Research Service, Department of Veterans Affairs.
Alexander Sloutsky is acknowledged for many helpful discussions. C. Fordham von Reyn and the Mycobacteriology Section of the Massachusetts State Laboratory, Boston, are acknowledged for their generous gifts of bacterial isolates.
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