INTERSFECIFIC HYBRIDIZATION: A POSSIBLE MODE OF THE ORIGIN I.
OF "ATYPICAL ACID FAST MYCOBACTERIA.

In a preliminary experinent a strain of chromogenic acid fast
mycobacterium was obtained by the mixed cultivation of a BCG strain
of M. tuberculosis and a saprorhytic mycobacterium: M. phlei *
Colonies of the new organism iigesiidbbdddiGesRNeie. were picked froma
selective mdium, that contained 50 ug streptomycin per ml and
tryptose agar and which did not allow the growth of either of the
parent organisms if planted alone.
The streptomycin resistance /s¥ / of the BCG strain, on the ore hand,
the pigment formation / Pig*/ and the capability of growth on
plain agar medium /Agt/ of M. phlei, on the other hand , were selected
as marker characteristics /BCG = S™ Pig” Ag , M. phlei = SS pig* apt/,
Other differences in the properties of the parent strains like: growth
at room temperature, in liquid medium, peroxydase activity, resistance
to INH, PAS, colony appearance and cellular morthology have added to
the number of distingishing features as unselected markers /See Plate I/
The descendant WWM organism: S’Pig* ag*t retained a mch larger
portion of the main features of M. thlei than tose of M. tuberculosis
/soG/, as for instance the capability of growth on agar, pigment

formation, colony appearance etce

 

* By a slight modification of the experimental conditions 8 more
strains were obtained, but could not yet be examined in all details.
A prolonged cultivation of the culture mixture in a nedium favoring

growth of both parénmt organisms prior to the exposure to the
selective environment was found to yield a more uniform result.
Te

It did inherit on the other hand the streptomycin resistance of the BOG
parent organism, displayed it, though at a much higher level than
it was established in the BCG strain / resistance to over 1000 ug per
ml as compared to 50 ug per ml: 20 fold increase/. In addition to
this considerable increase in the streptomycin mesistance Wa@inu@ttis of
hayigiden, showed on first isolation another new feature: a sort of
streptomycin semi-dependance which presented itself in the better
and faster growth of the micro-organism in the presence of streptomycin
both at 37° ¢ and at room temperature. Apart from the emergence of
such #. new properties, and t. properties which resembled those poss-
essed by one or the other of the parent strain, or even both of them/
e.g. catalase activity / ’ % a considerable number of characters app-
eared kind of inbbetween the properties of the BOG and M, phlei
strain / e.g. INH, PAS resistance, peroxydase activity, growth in
liquid medium, at room temperature /.

A population study showed evidence that segregation in the sew
Wel organism occurred at a very high rate when subcultures were
made on non-selective medium,- segregants mainly resembled the M.
thlei parent organism. Only a minor portion of the population retained
after 15 passages on non-selective medium the streptomycin resistance,
mit even this portion of the population showed properties otherwise
similar to those of M. phlei / INH and FAS resistance, growth at
room temperature ect. / Streptomycin-dependant individuals were, of
course, selectively eliminated of the population in the absence of
streptomycin. - If , however, serial transfers were made on media

containing streptomgcin, not only did the selective environment favor
Ite
the outgrowth of streptomycin resistant, and the more so the streptom-
ycin-de pendant individuals, but helped at the same time to conserve
some of the original traits of the new strain / €ege growth at
room temperature /.- Pure «clone studies are on the way.

The clumpy growth of mycobacteria is well Imow to cause
difficulty in making a reliable viable cell count, still a rough
estimation would show that the rate of occurrence of the gig
organism is somewhat in the proximity of 10° - 107° counted per
M. phlei cells

No systemtical attempt was as yet made at excluding the
possibllity of transformation or transduction as the genetic mechanism
by which the individuals of the new strain are produced. Still, there
exists some indirect cytological evidence indicating that a cell-to-cell
contact: i.e. conjugation may take place and that this could play a
major role in their origin. While the change from the streptomycin
sensitivity to the streptomycin resistance per se could te attrimted
to an additional mtational step, the assumption that mtation alor,
as a mere change from the streptomycin sensitivity of the M. thlei
organism to the streptomycin resistance may wxplain the whole phen-
omenon seems unlikely in view of

I. the high frequence of segragation,

2. streptomycin alone cold never bring about changes
in the M, phlei strain as experienced with large numbers
of controls / although the possible inducing role of
the BOG organism in the presence of streptomycin at
first could not be completely discounted /.

3. finally- and this appears to be of decisive importance -
“it could be shown that the mixed cultivation in a
streptomycin free medium of a/ the streptomycin sensitive

M. fhlei strain, which has never been in any previous
contact to streptomycin, and b/ the streptomycin resistant
M. tuberculosis / BCG / strain, resulted in the formation

of chromogenic strains completely resistant to streptomycin,
when plated on tryptose agar containing 500 ug streptomycin
per ml,

Conclusion: A new strain of mycobacterium: Iséetupttsdoun
Welle was obtained by mans of a mixed cultivation of

M. tuberculosis/ BCG / and M. thlei. The progeny appears

to possess som new characters, while others resemble

the characters of the two parent organisms. The main
interest in obtaining such strains lies in the fact

that the properties of thes Sides. organism are character-
istic for the " atypical " acid fast chromogenic myco~
bacteria, the origin of which aroused such a big interest

in recent yearse
SM resistance
over £ ug
over X00 ug Fer ml

Pigment formation
color of pignent

Growth on agar
Growth in Tween-alb.
Growth at room temp.
days required
on agar
on SM agar /0ug/
INH resistance
over lug ..
over 10 ug Per ml
FAS resistance
over Tug pr ml.
over 100 ug
Gatalase activity
Feroxydase activity
Golony appearance

Cellular morpology

* Unstable characters.

TABLE I.

PARENT _
Me. tb. / Boa /

Deposit

+
R

Mainly slender
acid fast rods

STRAINS
M. phiei

+

Turbidity,
later deposit

+

7-15

I+ +

s

Pleomorhic forms
Many coccoids.
Partial acid fast
-ne ss

Ve

NEW STRAINS

+
+ / T9000 ug/
+

yellow which
can change to
orange

Pi

Mainly deposit
first

+ /slow/

20=25%
5-12

git

i+

8

Ple omorthisme
forms varying
with culture
me diume