Geographic Distribution of Phage Types among Cultures of Mycobacterium tuberculosis II. Cultures from India and South Africa 1•2

WILBUR D. JONES, JR.

Introduction

In the past several years, we have developed new methods for phage-typing Mycobacterium tuberculosis cultures (1, 2), and we have successfully applied phage-typing techniques to cultures obtained during tuberculosis outbreaks (3) and suspected laboratory cross-contamination incidents (4) and to those from widely separated geographic areas (5). The phage-typing of M tuberculosis cultures from selected geographic areas was undertaken to determine (1) if these different geographic areas have one or more unique or predominant phage types, and (2) if the 10mycobacteriophages now used for typing are sufficient to type M tuberculosis cultures from different areas of the world. In the present report, we describe the phage-type distribution patterns of M tuberculosis cultures from India and South Africa and compare the phage-type distribution with that of cultures from the United States and Southeast Asia. The potential value of phagetyping to measure the impact of infection by immigrant populations on the extent and types of tuberculosis found in the United States is discussed. Methods Mycobacteria The 74 M. tuberculosis strains from Madras, India, were obtained from Dr. S. P. Tripathy, Director, Thberculosis Research Center, Madras, India. The 78 M tuberculosis strains from South Africa were obtained from Dr. H. H. Kleeberg, Tuberculosis Research Institute Pretoria, South Africa. ' Mycobacteriophages and Phage Typing The 10 mycobacteriophages used for typing their indicator host strains, and the method~ for the phage-typing were described previously (1, 2). The major phage-type designations 1to 8 are based on the lytic patterns produced by the Mycobacterium typing human phages (MTPH) 2, 3, 4, and 5, whereas the sixMTPH 1000

SUMMARY Mycobacterium tuberculosIs cultures obtained from India and South Africa were phagetyped to determine distribution patterns according to phage type In these two geographic locations. Of the 74 Indian strains, 14.9% were type 1, 32.4% were type 2, 28.4 were type 7, and 24.3% were type 8, whereas of the 78 South African strains, 20.5% were type 1, 47.5% were type 2,1.3% were type 4, 11.5% were type 7, 19.2% were type 8. The phage types were then subdivided according to the lytic patterns produced by the six auxllary phages. The phage type distribution In the Indian and South African strains was compared with the type distribution In cultures from the United States and Southeast Asia, and differences were found In the four widely separated geographic areas. AM REV RESPIR DIS 1990; 142:1000-1003

auxiliary phages 7, 9, 12, 13, 14, and 15 are used to further subdivide each major phage type (2, 3).

Results

The distribution of major phage types of M tuberculosis in the different geographic locations is listed in table 1. All 74 Indian strains were of phage types 1 (14.9070), 2 (32.4070), 7 (28.4070), and 8 (24.3070). The 78 South African cultures were of phage types 1 (20.5070), 2 (47.5070), 4 (1.3070), 7 (11.5070), and 8 (19.2070). The one South African strain of phage type 4 is the only one of this type found among the 700 cultures tested from the four geographic areas. The Indian cultures contained a larger proportion of phage type 7 strains (28.4070) than was found among cultures from South Africa (11.5070) or the United States (1.3070). No type 7 strains were identified among the Southeast Asian cultures. No phage type 5 strains wereobserved in the Indian or South African isolates, whereas 23.2070 of the Southeast Asian cultures and 2.6070 of the U.S. cultures had strains identified as phage type 5. Each of the major phage types may be further subdivided by the lytic patterns produced by the six auxiliary phages, MTPH 7, 9, 12, 13, 14, and 15. The lytic patterns produced by the auxiliary phages have been helpful in tracing epidemic strains; therefore, the distribution of auxiliary phage type subgroups is presented

in table 2 for India and South Africa. Some auxiliary lytic patterns in each phage type were similar among the cultures from the four geographic areas, whereas other auxiliary patterns were unique to, or more predominant in, one geographic area than in another. For comparative purposes, the proportions of the auxiliary phage patterns within each major phage type in the geographic areas studied are listed in table 3. Discussion

Investigations of the bacteriophage types of Mycobacterium tuberculosis cultures from widely separated geographic areas were undertaken to determine (1) if one or more phage types wereunique to a particular area, (2) if the four major and six auxiliary phages were sufficient to type (Received in original form February 6, 1990 and in revised form May 30, 1990) 1 From the Mycobacteriology Laboratory Respiratory Diseases Branch, Division of Bac;erial Diseases, Center for Infectious Diseases Centers for Disease Control, Public Health Ser;ice, U.S. Department of Health and Human Services, Atlanta, Georgia. 2 Correspondence and requests for reprints should be addressed to Wilbur D. Jones, Jr., MycobaeteriologyLaboratory,RespiratoryDiseases Branch, Division of Bacterial Diseases Center for Infectious Diseases, Centers for Disea'se Control Public Health Service, U.S.Department of Health and Human Services, Atlanta, Georgia 30333.

GEOGRAPHIC DISTRIBUTION OF ",. TUBERCULOSIS

1001

PHAGE TYPES

cultures from different areas of the world or whether additional phages were needed, and (3) if a general baseline of phage type distribution would be valuable for epidemiologic investigations. Our studies have addressed each of these points. Perhaps the most nearly unique phage type is type 5, which accounts for nearly one-fourth of all Southeast Asian phage types (table I). However, this phage type is not found in any of the other geographic areas studied to date except the United States. The verylow percentage (2.6070) of phage type 5 M tuberculosis cultures in the United States may be due to (1) a low level natural occurrence or (2) a dissemination of this phage type by earlier Asian immigrants to this country or (3) both. Whatever the reason, a sudden increase in patients with phage type 5 M tuberculosis newlydiagnosed in India, South Africa, and even the United States would strongly suggest a point source case(s) in (or in someone infected by) a Southeast Asian immigrant(s). Similarly, an increase in cases of tuberculosis phage type 7 among Southeast Asians or even native-born residents of the United States could suggest infection by immigrants (or visitors) from India or South Africa. The four major phages and the six auxiliary phages that we now use for typing strains of M tuberculosis seem to be a sufficient number of phages to type cultures from any geographic area of the world. We have not yet encountered any pure cultures of M tuberculosis that we were unable to type. If a culture was not lysed by phage MTPH 2 (DS6A), further studies demonstrated the culture to be either a mixed culture of M tuberculosis with another species of mycobacterium or not M. tuberculosis. Although all M. tuberculosis cultures could betyped with the four major phages (MTPH 2, 3, 4, and 5), the subdivision into only eight major phage types is not sufficient for use in either geographic or epidemiologic studies. The addition of the lytic patterns produced by the six auxiliary phages further subdivides strains in each phage type, which yields a possible 64 subtypes for each major phage type (3). In an earlier investigation, we performed repeat tests on a number of cultures and found no indication ofvariability in the phage type or in the lytic patterns produced by the auxiliary phages (2). We found strains in each phage type from different geographic areas that had aimilar auxiliary phage patterns (table 3), :'Nhereas other strains within each phage

TABLE 1 PHAGE TYPE DISTRIBUTION AMONG MYCOBACTERIUM TUBERCULOSIS CULTURES FROM INDIA AND SOUTH AFRICA * Indian Strains

South African Strains

Southeast Asian Strainst

United States Strainst

Phage Type

(n)

(%)

(n)

(%)

(n)

(%)

(n)

(%)

1 2 3 4 5 6 7 8

11 24 0 0 0 0 21 18

14.9 32.4 0 0 0 0 28.4 24.3

16 37 0 1 0 0 9 15

20.5 47.5 0 1.3 0 0 11.5 19.2

9 49 0 0 20 0 0 8

10.5 57.0 0 0 23.2 0 0 9.3

157 194 0 0 12 0 6 93

34.0 42.0 0 0 2.6 0 1.3 20.1

74

100.0

78

100.0

86

100.0

462

100.0

Total

• For comparative purposes, the phage types of cultures from Southeast Asia and the United States are also listed. t Data adapted from previously published studies (2, 5).

type had auxiliary patterns that were parent phage D34·F-130, which identifies phage type C strains, these investigators found in only one area (table 2). In earlier phage-typing investigations, used phage D34·LR-14 to designate phage Bates and Fitzhugh (6) used mycobac- type C strains and reported 73 (17.3070) teriophages D34·F-130 to designate phage of their 422 cultures to be phage type C type C strains. Phage type C M tuber- strains. These data confirm our obserculosis cultures were lysed by all the typ- vations of the expanded host range of ing phages just as phage type B strains phage D34· LR-14 and further demonwere, but type C strains were also lysed strate that phages D34·F-130 and D34· by phage D34·F-130(MTPH 6), whereas LR-14cannot be interchanged to identiphage type B strains were not. Also, fy phage type C strains, as originally dephage type C strains were reported to be scribed by Bates and Mitchison (7). The rare and to be restricted primarily to cul- results of the present study also confirm tures isolated from patients in the West- the broader host range of phage D34· ern Hemisphere (7). In our search for a LR-14 (MTPH 15)to lysestrains of differmore stable and reproducible phage D34, ent phage types (table 2). We reported a new phage type, type we evaluated phage D34·LR-14, which had been used by Rado and coworkers 7, in earlier studies of cultures from the (8) in the investigation of modification- southeastern United States (10). There restriction among M tuberculosis cul- were no phage type 7 cultures among the tures. We found phage D34·LR-14 to be Southeast Asian cultures, and among the more stable than the parent phage D34· United States cultures only 1.3070 were of F-130; however, we also found phage this type (2);however, 11.5070 of the South D34·LR-14(unlike the parent D34·F-130) African cultures and 28.4070 of the Indito have a broader range; i.e., it also lysed an cultures were phage type 7 (table I). strains in phage types other than phage One phage type 4 culture was identitype C strains. Because parent phage fied among the South African cultures, D34· F-130was found to be unstable, and and we found type 4 cultures in our earphage D34·LR-14 was more stable and lier evaluationof the typing phage MTPH possessed a broader host range, we rec- 13 (10); however, we did not find type 4 ommended phage D34·LR-14 as MTPH strains in either the Indian (table I), 15 and suggested that the use of parent Southeast Asian (5), or United States culphage D34·F-130 (MTPH 6) be discon- tures (2). To date, we have found only one tinued (2). The substitution of D34· type 6 culture (10)and one type 3 culture LR-14 for the parent phage D34·F-130 (Jones, WD, unpublished observations). would mean that phage type C strains Our data indicate that although strains of M tuberculosis, as described by Bates of major phage types 3, 4, and 6 are rare, and Fitzhugh (6) and Bates and Mitchi- these types do occur. son (7), could no longer be identified. Phage typing is a valuable adjunct to Recently, Clavel-Seresand Clement (9) epidemiologic investigations of tubercuused the phage-typing designation of A, losis outbreaks. We use our geographic B, C, and I of Bates and Mitchison (7) phage type distribution studies for basefor M tuberculosis strains from Europe, line information; however, each tuberAfrica, and India. Instead of using the culosis outbreak must be considered an

2

Phage Type

1 1 1 2 2 2 2 2 2 2 2 2 1 1

2 2 2 2 1 2 2 2 2 2 2 2 2

1 1 1 1 2 2 2 2 2 2 2 2 2

1 1 1 1 1 1 1 1 1 1 1 1 2 2

9

7

1 1 2 1 1 1 1 1 2 2 2 2 1 2

1 1 2 2 2 1 2 2 2 2 2 2 2

12

1 1 1 1 1 1 1 2 1 1 1 2 1 1

1 2 1 2 1 1 1 1 1 1 2 2 2

13

Auxiliary Phage Patternst

-

Phage Type

1 2 2 1 1 2 2 1 1 2 2 2 2 2

1 2 2 2 2 2 1 1 2 2 1 2 2

14

1 1 2 1 2 1 2 2 1 1 2 2 1 2

2 1 2 2 2 2 1 2 1 2 2 1 2

15

1 2 3 0 0 1 0 0 2 1 1 1 1 3

1 1 1 0 1 0 1 1 2 1 0 1 1

India

1 0 0 10 9 5 1 4 0 1 0 1 0 0

3 0 1 2 0 1 0 1 1 2 3 0 1

South Africa

7 9

1 1 1 1 1 1 1 2 2 2 2 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 2 2 1 1 1 1 1 2 2 2

7

8

1 1 1 1 1 2 2 2

1 1 1 1 2 2 2 1 1 1 2 2 2

2 2 2 2 2 2

1 1 1 1 2 1 1 1

1 1 1 2 1 1 1 1 1 2 1 1 1

1 1 1 1 1 2

13

1 1 2 2 1 1 1 2

1 2 2 1 1 1 2 1 2 1 2 2 2

2 1 1 2 2 2

14

t 1 = lysis; 2 .. no lysis.

* The number of strains found with each auxiliary phage pattern is listed also.

1 2 2 2 2 2

2 2 2 2 2 2

12

Auxiliary Phage Patternst

Human Mycobacterial Typing Phage Number of Strains with Each Auxiliary Phage Pattern

Human Mycobacterial Typing Phage

TABLE 2 CONTINUED

TABLE 2

LYTIC PATTERNS PRODUCED BY THE SIX AUXILIARY PHAGES WITHIN EACH PHAGE TYPE IN THE MYCOBACTERIUM TUBERCULOSIS STRAINS FROM INDIA AND SOUTH AFRICA *

8 1 0 1 1 1

0

2 1 1 2 2

6

1 0 1 1 2 0 0 0 1 1 2

6 6

3 3 1 1 3 0

India

1 2 1

1 1 2 1 1 2 1 2 1 2 1 1 2

2 1 2 1 2 2

15

5 1 7 0 2 0 0 0

0 1 0 1 1 0 0 2 2 1 1 0 0

0 2 0 1 0 2

South Africa

Number of Strains with Each Auxiliary Phage Pattern

c;II

JJ

z

cO

P

:II

~ rc: III

N

o

.... o

GEOGRAPHIC DISTRIBUTION OF M. TUBERCULOSIS

1003

PHAGE TYPES

TABLE 3

References

AUXILIARY PHAGE PATTERNS OF INDIAN AND SOUTH AFRICAN MYCOBACTERIUM TUBERCULOSIS·

1. Jones WD Jr, Greenberg J. Modification of methods used in bacteriophage typing of Mycobacteriumtuberculosis isolates. J Clin Microbiol1978; 7:467-9. 2. Jones WD Jr, Good RC, Thompson NJ, Kelley GD. Bacteriophage types of Mycobacterium tuberculosis in the United States. Am Rev Respir Dis 1982; 125:640-3. 3. Snider DE Jr, Jones WD, Good RC. The usefulness of phage typing Mycobacterium tuberculosisisolates. Am Rev Respir Dis 1984;130:1095-9. 4. Jones WD Jr. Phage typing of Mycobacterium tuberculosis cultures from incidentsof suspected laboratory cross-contamination. Thbercle 1988; 69:43-6. 5. Jones WD Jr, Woodley CL. Phage-type patterns of Mycobacteriumtuberculosis from Southeast Asian immigrants. Am Rev Respir Dis 1983; 127:348. 6. Bates JH, Fitzhugh JK. Subdivision of the species Mycobacterium tuberculosis by mycobacteriophage typing. Am Rev Respir Dis 1967; 96:7-10. 7. Bates JH, Mitchison DA. Geographic distribution of bacteriophage types of Mycobacterium tuberculosis. Am Rev Respir Dis 1969;100:189-93. 8. Rado TA, Bates JH, Fitzhugh JK. Evidence for host-dependent modification and restriction of bacteriophage DNA in Mycobacterium tuberculosis. Am Rev Respir Dis 1978; 118:835-42. 9. Clavel-Seres S, Clement F. Repartition des lysotypes de Mycobacterium tuberculosis en relation avec le pays d'origine du malade. Ann Inst Pasteur Microbiol 1984; 135:35-44. 10. Jones WD Jr, Greenberg J. Use of phage FOWJ-lof Mycobactenum fortuitum to discern more phage types of Mycobacterium tuberculosis. J Clin Microbiol 1976; 3:324-6.

Human Mycobacterial Typing Phage Number of Strains with Each Phage Pattern in Each Geographic Area Phage Type

2

Auxiliary Phage Patterns t

7

9

12

13

14

15

India

South Africa

Southeast Asia*

United States*

1 1 1 2 2 2

1 2 2 2 2

2 2 2 2 2 2

1 1 2 1 1 2

1 2 2 1 2 2

1 2 2 2 2 2

2 1 0 1 1 1

1 1 2 1 2 1

3 0 1 2 1 3

0 8 11 5 26 60

1 1 1 1 1 2 2 2 2 2

1 2 2 2 2 1 2 2 2 2

1 1 1 1

1 1 1 1 2 1 1 1 1 2

1 1 2 2 2 2 1 1 2 2

1 1 1 2 2 2 1 2 2 2

1 0 1 0 2 3 3 1 3 0

1 10 5 1 2 0 2 0 0 2

1 1 0 1 1 2 0 5 9 16

0 6 3 22 1 5 1 1 33 25

6 8

5 7

3 0

41 15

2

2 2 2 2 2 2

1 2

8

• For comparative purposes, the number of strains from the United States and Southeast Asian cultures that had similar auxiliary phage patterns is listed. t 1 lysis; 2 no lysis. :j: Data adapted from previous studies (2, 5).

=

=

independent entity, and unrelated, but coded, cultures of M tuberculosis should always be submitted from the community along with the coded cultures suspected to be epidemiologically related. This procedure (1) indicates whether or not

the community has a predominant phage type and (2) helps eliminate any unintentional bias in the phage typing since the relationship of the coded cultures is unknown to laboratory personnel.