Most Human Isolates of Mycobacterium avium Mav-A and Mav-B Are Strong Producers of Hemolysin, a Putative Virulence Factor

Abstract

Hemolysin was quantified in 58 isolates of Mycobacterium avium from human, animal, and environmental sources. Human Mav-A and Mav-B isolates were the strongest producers; in contrast, animal and environmental Mav-A isolates and human, animal, and environmental Mav-C organisms were low-level producers. Hemolysin production was not restricted to isolates causing invasive infections.

Mycobacterium avium, one of the organisms of the M. avium complex, behaves as an opportunistic human pathogen. The organism can be isolated from environmental, animal, and human sources, and tap water is regarded as the main reservoir of the organism (17). In immunocompetent patients, M. avium causes (although rarely) pulmonary infections and cervical lymphadenitis and, occasionally, soft-tissue infections. During the early years of the AIDS epidemic, M. avium caused severe disseminated infections in a high proportion (25 to 50%) of severely immunocompromised AIDS patients (5, 10). The development of the highly active antiretroviral therapy (HAART) has greatly reduced morbidity and mortality caused by M. avium infection in AIDS patients (1, 15), but nowadays, the organism still represents a threat to newly diagnosed patients and to patients who cannot afford HAART or for whom HAART is ineffective.

M. avium strains can be typed into seven sequevars (named Mav-A to Mav-G) on the basis of the nucleotide sequence of the 16S to 23S rRNA gene internal transcribed spacer (ITS) (4, 6, 13). Organisms of sequevar Mav-A have been isolated from HIV⁺ and HIV⁻ patients, as well as from environmental and animal sources, while Mav-B organisms are associated almost exclusively with severe disseminated infections in AIDS patients or with lymphadenitis in infants; other ITS subgroups appear to be extremely rare in human infections (4, 7, 8, 14). The sequevar-related differences in the epidemiological characteristics of M. avium infections make it plausible that different M. avium sequevars have different virulence properties.

The mechanisms by which M. avium determines infection and disease in humans are poorly known, and several attempts have been made to define specific markers for M. avium clones pathogenic for humans. Only a few properties of the organism have been associated with virulence so far; for example, variations in the virulence of M. avium isolates often correlate with differences in colony morphology (2), in the serovar (16), in certain genetic markers (12), or even in as-yet-unknown host-range factors (3). It was reported a few years ago that invasive isolates of M. avium causing disseminated infection produce a magnesium-dependent, cell wall-associated hemolysin and that the large majority of isolates from localized noninvasive pulmonary infection are nonhemolytic (11), which suggested that hemolysin represents a virulence factor necessary for invasive disease.

In this study, we have quantified hemolysin production in 58 M. avium strains, including 27 human isolates, 25 animal isolates, and 6 environmental isolates from the Pisa geographic area. The human strains were isolated in our laboratory by the radiometric BACTEC system (Becton Dickinson) from clinical specimens; the animal and environmental isolates were submitted to our laboratory from the Veterinary Department of the University of Pisa for reference purposes. The animal strains were from swine (13 isolates) and birds (12 isolates from six different aviary species), and the environmental isolates were from stream water. All the isolates were identified by molecular probes (Gen-Probe) and typed according to a previously described IS1245-based assay (14) (see below). After primary culture isolation, the isolates were subcultured in Middlebrook 7H9 medium and aliquots of cultures were frozen at −80°C until being used for the present study. The isolates were typed for the ITS subgrouping as described by Frothingham and Wilson (6), with slight modifications (14).

For the hemolysis assay, the organisms were grown in 7H9 broth until an absorbance value of 0.2 at 600 nm was achieved. Bacteria were then washed once, resuspended in Alsever's solution to a concentration of 5 × 10⁹ CFU/ml, and immediately assayed. The hemolytic assay was performed as described by Maslow et al. (11), with slight modifications. Briefly, hemolysis was determined in duplicate in 5-ml sterile polystyrene snap-top tubes containing 3 ml of Alsever's solution, 10 mM CaCl₂, 10 mM MgSO₄, 25 μl of washed, defibrinated sheep erythrocytes (Oxoid), and 5 × 10⁸ CFU of bacteria. Sample tubes were incubated for 6 h at 37°C and then gently mixed, and the erythrocytes were pelleted at 700 × g for 10 min. Tubes containing no bacteria were included as controls. The absorbance at 520 nm (A₅₂₀) of 1 ml of supernatant was determined spectrophotometrically (UltraSpec; Pharmacia) in glass cuvettes. A strain was considered hemolytic when the A₅₂₀ in the presence of tested bacteria was above the mean value plus 2 standard deviations (SD), as measured for control tubes containing no bacteria.

The hemolytic activity of the isolates was quantified according to the ratio between the mean absorbance values obtained in the presence (A₅₂₀⁺) and in the absence (A₅₂₀⁻) of tested bacteria. The level of hemolysis was also expressed in a semiquantitative way as follows: an A₅₂₀⁺/A₅₂₀⁻ value of <1.5 signified weak hemolysis, a value between 1.5 and 2.0 signified moderate hemolysis, and a value of >2.0 signified strong hemolysis. In our hands, the use of the hemolytic ratio instead of the net absorbance values used by Maslow et al. (11) allowed the highest reproducibility of the results by reducing day-to-day variation due to slight differences in the spontaneous hemolysis of the erythrocyte preparations used for each assay.

As shown in Table 1, 33 isolates were of sequevar Mav-A; of these, 11 were isolated from humans (6 and 5 isolates from HIV⁺ and HIV⁻ patients, respectively), 20 were isolated from animals (including the aviary reference strain M. avium ATCC 35712), and 2 were isolated from water. Of the 15 Mav-B organisms, 14 were from HIV⁺ patients. In contrast, only one isolate of Mav-C was from a HIV⁺ patient, while 5 and 4 Mav-C isolates were from animal and environmental sources, respectively. All human Mav-A organisms showed distinct multibanded IS1245-restriction fragment length polymorphism (RFLP) profiles (data not shown), although two clusters of 3 and 2 highly similar isolates (similarity coefficients, 96.3% and 97.7%, respectively) were detected. Similarly, the 15 human isolates of Mav-B yielded 10 individual multibanded IS1245-RFLP patterns, one cluster of 2 highly similar isolates (similarity coefficient 97.0%), and one cluster of 3 identical isolates; the only human isolate of Mav-C yielded a distinct multibanded IS1245-RFLP profile. In contrast, the IS1245-RFLP profiles of the animal and environmental isolates of Mav-A and -C, including the aviary reference strain, presented a double-band RFLP profile, which does not allow differentiation of the strains.

TABLE 1.

Hemolysin production by isolates of M. avium in relation to the source and ITS typing

Isolate source and sequevar	Total no. of isolates (no. of isolates from HIV+ patients)	Results for hemolysin production
		No. of strains with the indicated level of hemolysisa				A520+/A520− ratio (mean ± SD)b
		None	S	M	W
Human
Mav-A	11 (6)		10	1		2.54 ± 0.45
Mav-B	15 (14)	1	12	1	1	2.56 ± 0.61
Mav-C	1 (1)			1		1.64
Total	27 (21)	1	22	3	1	2.52 ± 0.57
Animal
Mav-A	20	2	2	12	4	1.69 ± 0.33
Mav-C	5			4	1	1.63 ± 0.07
Total	25	2	2	16	5	1.68 ± 0.30
Environmental
Mav-A	2				2	1.30 ± 0.22
Mav-C	4	3			1	1.15
Total	6	3			3	1.25 ± 0.18

^aS, strong; M, moderate; W, weak. None, not hemolytic.

^bMeans ± SD of the ratios between the absorbance values obtained in the presence (A₅₂₀⁺) and in the absence (A₅₂₀⁻) of tested bacteria. Only hemolytic strains were considered.

Of the 58 strains tested, 52 showed hemolytic activity, although to different extents. Nonhemolytic strains were as follows: one Mav-B human isolate, two Mav-A animal isolates, and three Mav-C environmental isolates. Analysis of variance showed statistically significant differences in levels of hemolysin production among the sequevars (P = 0.0004) as well as among the organisms of different sources (P < 0.0001). Most Mav-B organisms were equally strong producers (mean ± SD of A₅₂₀⁺/A₅₂₀⁻ ratios of 14 hemolytic strains, 2.56 ± 0.61); on the other hand, the seven hemolytic Mav-C organisms were moderate or weak producers (mean ± SD of A₅₂₀⁺/A₅₂₀⁻ ratio, 1.56 ± 0.19 [P < 0.001 and P < 0.05 versus Mav-B and Mav-A organisms, respectively]). Mav-A organisms of human origin produced the same amounts (mean ± SD, 2.54 ± 0.45) of hemolysin as Mav-B isolates, while Mav-A animal and environmental isolates were significantly less hemolytic (means ± SD, 1.69 ± 0.33 and 1.30 ± 0.22 for animal and environmental isolates, respectively [P < 0.01 at least]).

When hemolysin production by human isolates was considered in relation to that of clinical specimens (Table 2), we found that all the respiratory isolates, as well as all but one isolate from blood and other specimens, were hemolytic; the level of hemolysis was generally high. The finding that hemolysin production was not restricted to isolates causing invasive infections does not confirm the report of Maslow and colleagues (11), according to which only 1 (7%) of 15 M. avium respiratory isolates in their study was hemolytic. Nonetheless, our observation does not scale down the role of hemolysin in human infection, as we found that hemolysis expression is almost universal among human isolates and is particularly high among the organisms of the Mav-A and -B sequevars, i.e., those that cause the vast majority of human infections. Host factors, such as severe immunodepression, likely play a critical role in allowing hemolytic organisms to gain entry to the bloodstream and cause invasive disease.

TABLE 2.

Hemolysin production by human isolates of M. avium in relation to clinical specimen, sequevar, and patient's HIV status

Clinical specimen and sequevar	No. of isolates from HIV+ patients/total no. of isolates	Results for hemolysin production
		No. of strains with the indicated level of hemolysisa				A520+/A520−b
		None	S	M	W
Blood
Mav-A	2/2		2			2.48 ± 0.66
Mav-B	14/14	1	11	1	1	2.51 ± 0.69
Mav-C	1/1			1		1.64
Pulmonary
Mav-A	1/5		5			2.77 ± 0.46
Mav-B	0/1		1			2.27
Otherc
Mav-A	3/4		3	1		2.28 ± 0.27

^aS, strong; M, moderate; W, weak. None, not hemolytic.

^bMeans ± SD of the ratios between the absorbance values obtained in the presence (A₅₂₀⁺) and in the absence (A₅₂₀⁻) of tested bacteria. Only hemolytic strains were considered.

^cSpecimens other than blood and pulmonary samples included urine (two specimens), cerebrospinal fluid, and stool samples.

The mechanism by which hemolysin may contribute to successful infection in humans has to be clarified. It should be emphasized, however, that hemolysins are likely important components of mycobacterial pathogenicity. In fact, the presence of a membrane-active hemolysin which lyses cells through pore formation (9) and which has a high level of amino acid homology to the TlyA hemolysin from the swine pathogen Serpulina hyodysenteriae has been demonstrated in Mycobacterium tuberculosis; a TlyA homologue has also been identified in M. avium (18). On the other hand, the predominance in human infections of Mav-A and Mav-B strains that produce a high level of hemolysin might also reflect their selection or adaptation to a particular environmental niche that facilitates diffusion to humans. The search for determinants directly involved in M. avium pathogenicity will likely contribute to define specific markers for pathogenic clones and to clarify many aspects of the epidemiology of M. avium infections.