gram-negative-bacilli isolated from the sputum in …...five-bacilli isolated from the sputum, tyre...
TRANSCRIPT
[GRAM-NEGATIVE-BACILLI ISOLATED
\ FROM THE SPUTUM IN CASES OF H ASTHMA
By HUGH W. ACTON, c.i.e.
LIEUTENANT-COLONEL, I.M.S.
Professor oj Pathology and Bacteriology and Director
and
DHARMENDRA, m.b, b.s.
Assistant Research Worker, Calcutta School oj Tropical Medicine
Introduction.?Walker (1919) recovered a
Gram-negative-bacillus from the sputum of one out of 74 cases of asthma; the organism re-
sembled the coli type of bacilli in its sugar re-
actions. Rackemann (1920) investigating the
bacteriology of the sputum in 40 cases of asthma isolated a Gram-negative-bacillus in two cases.
Rackemann and Graham (1923) in the sputa
April, 1933] GRAM-NEGATIVE-BACILLI IN ASTHMA : ACTON & DHARMENDRA 193
from 29 cases of asthma found a Gram
tive-bacillus predominating 111 ,1 ma)
Thomas, Famulener .
and Touart (1924J
found a Gram-negative-bacillus (1926) Of 180 cases of asthma leported two eases ot astmii
treated with a vaccine made from
Eyre five-bacilli isolated from the sputum, tyre
(reported by Knott, 1929) special attention to the occurl ,, +
Gram-negative-bacilli and the testing
vaccine injection or intracutan ST3ecially patients could often be shown
o
bacillus sensitive to them. He found that
to ;bacUl^ grew well at 22?C. and called
it the
bacillus'. Knott, Oriel and examined isolated m 48 cases out of 132 -P tPOv>niaue, these Gram-negative-bacilli by d ar piates 1-e., incubating the spiitum
on b considered
at 22 C. Knott and Oriel (193U) t
that these bacilli might be produce many mine-like substance which is present m man.
asthmatic sputa. Significance of the
bacillus ^
(1) Histamine prodwe^on.?Knott, "eb Witts (1930) studied 22 strains
of thesed
in detail and found 10 strains to be h stamm^
Producers. Knott and Oriel (1930) various histamine-like effect from extract ̂
^ac_ asthmatic sputa. Referring back found tcnology of these particular sputa
y ^
that many showed in the Inonehia numerous Gram-negative-bacilli. usqtamine- culture extracts of these bacilli gave ^he bke effect similar to that obtaine
amine- ?Putum itself. They think that
the ^tarnm^ hke substance demonstrable m ti P
these We arisen as a result of the growth
ot tnes^ bacilli within the small bronchial tu , TTnrkavy connection it is interesting to note
lc a
(1930) had already shown the pre SpUta.
?^asm-producing substance in ast m c_ , ^on
Oriel (1932) considers that the local pioduictio
_
?f histamine in the bronchi in additi un(j_ }ng contraction of the plain muse e the ]ng the bronchi would also tend to
m uron_
Permeability of the epithelium lining th
chioles, and facilitate the entrance rofess0r
Proteins and possibly bacteria. Tnter-
^tcpowall (1932) speaking at the A^t to national Congress on Asthma drew
a +urous;h
this local production of bronchospasm the liberation of histamine in the bi
these bacilli. 7 . ^
(2) Cutaneous sensitivity to emvn
^danism.?Knott, Oriel and Witts (1930) iouna
that skin tests made with emulsion o
organisms gave positive results m 74 P
'
?f patients from whom they were iso a
,,
ln only 11 per cent, of patients in whom they
Avere absent. In cases tested for c ^ f
sensitivity to emulsions of cocci ?btal]1 vprp
their asthmatic plugs no positive reactio Stained.
(3) We have shown that in asthmatic cases with Gram-negative-bacilli infection there is
usually a very high blood eosinophilia and that they form a separate clinical group.
(4) Knott, Oriel and Witts (1930) found
eosinophils in the sputum in seven-eighths of his cases with Gram-negative-bacillus infection while only one half of other cases showed eosino- phils in the sputum, i.e., out of 48 cases with Gram-negative-bacillus 42 showed eosinophils in the sputum and 6 did not; of the remaining 84 cases only 43 showed eosinophilia in the
sputum and 41 did not. We do not find any correlation between
Gram-negative-bacillus infection and sputum eosinophilia; to this point we shall refer later.
In a series of 150 cases we obtained Gram-
negative bacilli in 27 cases, i.e., 18 per cent, of our cases.
Description of the bacillus
Morphology.?Non-sporing, short thick rods, straight or slightly curved, occurring singly, in
pairs or in small chains. Very pleomorphic in
cultures, when diplococcal or very long filamen- tous forms are seen.
Motility.?Non-motile. Staining reactions.?Gram-negative, non-
acid-fast, capsule not seen. Cultural characters.?Grows well on ordinary
media at our room temperature (27? to 32?C.) and at 37?C. Cultures kept in the cold-room (21? to 23?C.) did not grow. Gelatin stab (with 1 per cent, agar):?No
surface growth. Growth along stab. No liqui- faction.
Agar slant:?Fine opaque colonies with trans- mitted light, which coalesce together to make a streak. Growth moderately profuse.
Blood-agar slant :?No haemolysis, fine
transparent growth. Serum :?No visible growth. Smear shows
scanty growth. Nutrient broth :?Slight turbidity with a
watered-silk appearance, deposit at the bottom. Potato :?No visible growth, on staining short
diplococcal and bacillary forms are seen. Sugars :?No gas is formed in any of the
sugars tested, acid is usually formed in glucose, lsevulose, galactose, maltose, saccharose and
dextrin. Usually no acid is formed in lactose, dulcite, salicin and inulin. Mannite is usually fermented with the production of acid, but when the strain is kept for some time the sub- cultures do not attack mannite. Sugar reactions for the 15 strains studied are given in table I. Litmus milk :?Usually unchanged. Slight
acidity in one case and acid and coagulation in another?in this case lactose was also fermented with production of acid. Pathogenicity ?Non-pathogenic to mice, rats
and rabbits by intraperitoneal and intravenous injections. Heat.?Heating at 50?C. for 15 minutes kills
the organisms.
194 THE INDIAN MEDICAL GAZETTE [April, 1933
Histamine 'production.?This was studied in 8 strains with negative results. As we obtained
negative results with cultures in ordinary broth,
Table I.
A statement showing the sugar reactions and motility of 15 strains of Gram-negative-bacilli (reactions in lozvulose, galactose, arabinose, inulin and dextrin were studied in only 5
strains).
No 3 o g -2 S 3 ?? ? -3 ^
-s ? ^ s ? -S .? a ? - ^ ?jo S co S q ^ j ,_2 o <3 hS
1 - O A A A A 0 o As A A A O 2 OA A A A 0 O n 3 ? 0 A 0 A A 0 A n 4 0 A O A A O A O 5 ? OA AAA 0 0 O
OAAAAOOO. OA A A A O O O O A 0 O
8?00 As As As 0 O O A A O O 9? OA A A A O O O 10 ? OA A A A 0 O 0
\\ ? O A A A o O O O A A A O
J2? OA A A O A O O AOOO 13 ? OA A A O 0 O f) 14? OAAAOOOO 15 ? A A A A A A OAc
A = acid in sugar media. Ac = acid and coagulation.
As = slight acid. ? = non-motile.
O = no acid or gas in sugar media.
cultures made in serum broth and Martin's broth were also tested with negative results. When we come to compare the Gram-nega-
tive-bacilli isolated in our study to those isolated by others we note that :?
1. No description is given of the organisms except by Walker and Knott.
4 strains 1 strain 1 strain
2 strains 1 strain 2 strains
1 strain 1 strain 1 strain
7 strains 1 strain
Lactose
AG AG
Glucose
A A A
AG AG AG
A AG AG
AG AG
Table II.
Saccharose
AG
AG
Maltose
A AG AG
A AG AG
AG AG
Mannite
AG
AG AG
AG AG
Dul cite Lit?us milk
Alk. A A
A = acid. AG acid and gas. Alk. = alkaline.
A A A
A A A
2. The one strain isolated by Walker pro- duced acid and gas in the sugars undergoing
fermentation, and is allied to the coli type of
bacilli. No gas is produced in any sugar by
any of the strains studied by us.
3. The 22 strains studied by Knott showed a great variation as regards the sugar reactions* which are shown in a compressed form in
table II. His 22 strains can be divided into two groups
?non-lactose fermenters and lactose fermenters- Non-lactose fermenters can be further divided
into two sub-groups according to whether they produce acid (A) or acid and gas (A & G)
111
ApRiL, 1933] GRAM-NEGATIVE-BACILLI IN ASTHMA : ACTON & DHARMENDRA 195
glucose. Lactose fermenters can similarly be
divided according to whether they produce (A) 0r (A & G) in lactose.
LACTOSE
I I Glucose 0 Fermented
! I l I I I A AG A AG
6 strains 5 strains 3 strains 8 strains (1st sub-group) (2nd sub-group) (3rd sub-group) (4th sub-group)
For comparison we give the sugar
I11 a compressed table for the 15 strain V us (table III). Wp Cpp "fVinf ?
, (1) Fourteen strains out of the 15 are
non-
lactose fermenters. .
(2) The one lactose fermenter produces only
apid in lactose and other sugars, . -m;iar Knott's strains there is one strain tha 1
,
to this but differing in not fermenting sa
a^d mannite and in producing only aci . ..
^'hile our strain produces acid and coag
13
1 strain
2 strains ..
1 strain ..
6 strains ..
4 strains ..
1 strain
Lactose Glucose
Table III.
Maltose
As
Saccharose Mannite
As As
Dulcite Salicin Litmus milk
As
Ac
A = acid. As = slight acid. Ac = acid and coagulation. ?? = no reaction.
(3) Amongst the 14 ^"^^ttack'glucose there is one strain which does not sugars. and produces very slight acid m
o ^p^+prs
(4) The remaining 13 non-laetose f menters
Produce acid in glucose and belong ^ey sub-group in Knott's strains. Ho '
p as
differ from his 6 strains in this su
Wilder :? , _e
. (a) None of our strains produce a?7 Valine lT* litmus milk; 4 of his strains
make
ai*d two acid. ?wnse*
(b) All of our strains ferment sac
^one of his 6 strains attack this suga .
...
(c) Eight of our strains ferment man
n?ne of his six strains attack this sug ?
(d) Eleven of our strains ferment ina
?nly one of his strain ferments this sug ?, ^ (e) We have not been able to demonstrate
a capsule in any of our strains; Kno
Capsule in 2 out of 6 strains. , oo?P ? we
(/) All his strains grow well at *'
did not get any growth at 22? to 2o
It will be seen while our strains differ widely from Knott's, all of our strains resemble each other rather closely, thus leaving out the lactose-
fermenting strain and the one non-lactose-fer-
menting strain not fermenting glucose we find that :?
(a) All the 13 strains ferment glucose and
saccharose, and none of them has any action on milk.
(b) All but 2 ferment maltose and all but 5 ferment mannite.
(c) Dulcite and salicin are not as a rule
attacked; only one strain ferments dulcite and the two strains that do not ferment maltose ferment salicin.
Classification of the bacillus.?So that these 13 strains can be put together in the genus Eberthella under the tribe Bacteriese in the
family Bacteriacese. In its specific characters the organism is nearest to Eberthella phafii, the causative organism of fowl typhoid, being non-motile, a non-lactose and non-dulcite fer-
menter, forming acid in glucose, in saccharose and usually in mannite, and producing no change in litmus milk.
Its pleomorphic character makes it resemble
superficially Klebsiella pneumonias Fried- lander from which it differs in the following details :
(1) No capsule is seen.
(2) Growth not so abundant as in case of Klebsiella pneumonice.
(3) No gas is produced is any of the sugars fermented.
(4) Non-pathogenic to mice, rats and rabbits. Klebsiella pneumonice is highly pathogenic to mice; rabbits are less susceptible.
196
THE
INDIAN
MEDICAL
GAZETTE
[April,
1933
Table IV. Details of cases having Gram-negative-bacilli in sputum.
Serial num-
ber
Case number
10
11
12
17
26
29
30
31
36
38
40
52
57
58
65
Age and sex
34 F.
55 M.
38 M.
21 M.
18 F.
33 M.
38 M.
25 M.
Duration of illness
32 M.
30 M.
45 M.
30 F.
\
19 years
9 ?
10 ?
3 months
3 years
14 ?
4 ?
1 year
li years
H ?
9 ?
10 ?
Age at onset
15
45
28
21
15
19
34
24
30|
28 J
36
20
Effect of season or weather
Worse in summer
Worse in winter and rains.
Worse in winter
Pre-asthmatic state
Occasional colds
Severe bronchitis for 1 year pre- ceding.
Bronchitis 1 year
Blood count
Total leucocytes
Worse during the rains.
Running of nose
for some time. Irritation of nose with occasional bleeding. Pneu- monia 2 years before. Bron- chitis after
pneumonia. Pneumonia. Asthma started
just after pneu- monia.
Pneumonia 1 year before asthma.
Pneumonia, pleu- risy.
Pneumonia. Asthma started just alter pneu- monia.
12,000
7,000
13,400
16,000
25,400
16,000
8,000
25,000
16,000
16,000
16,000
10,000
Total
eosinophiles
960
420
6,618
9,600
5,350
5,760
1,360
13,500
9,400
6,720
3,000
800
Sputum eosinophiles
No.
No.
No.
A few eosino-
philes.
Some eosinophiles
No.
Eosinophiles make up 50 per cent,
of cellular exu- date.
Do.
No.
Treatment
Several philes.
No.
A u t o-v a c c i n e
sputum.
Do.
Not treated
A u t o-v a c c i n e
sputum.
Do.
Do.
A u t o-v a c c i n e
sputum.
Result
Do.
Relieved.
Much improved.
Relieved.
Do.
No improvement (old tuberculous lesion, right apex).
Not treated.
Much improved.
Improved.
Relieved. Had a
relapse 10 months later.
Not treated.
Do.
apri
l,
1933]
GRAM-NEGATIVE-BACILLI
IN
ASTHMA
: ACTON
&
DHARMENDRA
197
68 / 27 M. I 1 year j 26 j .. /Bronchitis 1 year I 9,000 I 6,000 I .. IA u t o-v a c c i n c j Improved. / / / / sputum.
77 42 M.
15 79
16 84
17 91
18 93
19 100
20 110
21 120
22 125
23 127
24 138
25 139
26 140
27 142
36 M.
28 M.
30 M.
20 M.
30 M
35 M.
32 M.
21 M.
42 M.
24 M.
36 M.
30 M.
52 M.
6 years
20
5
10
12 ?
6 months
10 years
8 ?
7 ?
1 month
6 years
5 ?
37 ?
36
16
23
20
18
18
34|
22
13
35
24
30
25
15
Worse in winter
Worse in winter
During rains only
During rains only
Worse in winter
In winter only
In extremes of
temperature.
Pneumonia 1 yeai before starting asthma.
Bronchitis for 5
years.
Tonsils removed at age of 5 and
again at 20.
Adenoids re-
moved at age of 12.
Chronic colds and sore throat.
Chronic bron- chitis.
Bronchitis for 3
years.
Pneumonia. Asthma started just after pneu- monia.
Bronchitis
17,000 5,000
8,000
16,000
10,000
9,000
8,000
7,000
6,000
15,000
11,000
7,000
25,000
15,000
7,000
800
10,240
1,000
4,000
2,000
700
1,440
6,000
1,000
700
16,000
7,000
770
Most of the cells j Do. ( Much improved, were eosino- phils.
Do.
No.
No.
No.
Eosinophils make up more than 50 per cent.
No.
No.
No.
No.
No.
No.
No.
3 doses auto- vaccine.
Aut o-v a c c i n e
sputum.
Do.
Do.
Aut o-v a c c i n e
sputum. Nose attended to.
Aut o-v a c c i n e
sputum. Hook- worm infection treated.
Aut o-v a c c i n e
sputum.
Aut o-v accine supplied.
Aut o-v accine
sputum.
Do.
Do.
Do.
Improved.
Not treated.
Relieved.
Improved.
Relieved.
Do.
Do.
Do.
Relieved.
Do.
Do.
Do.
Relieved = Completely relieved of symptoms. Much improved Intervals between attacks considerably increased, and the duration and acuteness of attacks lessened.
Improved Definite improvement but not very marked. Not improved = No improvement at all.
198 THE INDIAN MEDICAL GAZETTE [April, 1933
Before completing the description of the bacil- lus it may be worth while to refer to the few
experiments carried out with a view to observ- ing if the injection of the organism has any effect on the blood or tissue eosinophilia.
1. Broth culture of the organism injected subcutaneously in mice. Result?no local
eosinophilia. 2. Broth culture of the organism injected into
the pleural cavity of mice. Result?no local
eosinophilia. 3. Filtrate from the broth culture injected
intradermally in one patient (repeated injec- tions). Result?doubtful increase in the blood
eosinophilia, i.e., variations from none to 280
per c.mm.
A table detailing of the cases, with the Gram- negative-bacilli in the sputum is given.
Points from the table.
(1) Sputum eosinophilia. Out of 27 only 8 show eosinophiles in the sputum (out of the series of 150 cases 36 show eosinophilia of the sputum, and of the 79 cases of bronchial asthma not associated with Gram-negative-bacillus in- fection only 16 show eosinophiles in the sputum), so that cases with Gram-negative-bacillus in- fection have a higher incidence of sputum eosinophilia than other cases, but we have not found the incidence as high as in Knott's cases; he finds seven-eights of his cases showing sputum eosinophilia.
(2) High blood eosinophilia. This is not referred to by Knott, but in a personal com- munication he says
' all the patients infected with bacillary strains have shown eosinophilia (blood and sputum) or other manifestations of a'llergyThis high blood eosinophilia has been shown by us to be of diagnostic import- ance.
(3) Out of 27 cases 13 gave a history of in- heritance (in the total series of 150 cases only 40 gave a hereditary history, and out of 79 bronchial cases not due to these Gram-negative- bacilli, only 15 gave a history of inheritance).
(4) Age of onset. In 22 cases the age inci- dence varies from 11 to 30 years, and in 5 the
age of onset was above 30 years and of these 4 started within 36 years; there was only one patient who commenced his asthmatic attack at 45 years. (In 38 of the 79 bronchial cases without infection with the Gram-negative-bacil- lus the age of onset was above 30.)
(5) Effect of season. Out of 27 only 10 cases gave a history of seasonal variation and of these 7 were worse during the winter and rains or got the attacks only at this season, one was worse in summer, and two worse during extremes of temperature. (Of the 79 bronchial cases not
infected with the bacillus 35 shows seasonal variation and, of these 35, 29 were worse dur- ing the winter and rains, or got attacks only at those seasons.)
(6) Pre-asthmatic state. Of the 27 cases 18 or two-thirds gave histories of a previous illness,
i.e., 8 pneumonia, 7 chronic bronchitis, 2 nos and throat troubles and 1 dysentery. (Of ^ 79 bronchial cases without infection with tin
bacillus 52 gave a history of previous illness*
i.e., 16 pneumonia, 30 chronic bronchitis, 4 nose and throat, 1 dysentery and 1 urticaria. ^
the 22 gut allergic cases as many as 8 have a
history of previous dysentery?the important of the fact will be discussed in another paper-J A history of a previous pneumonia attack ^ more common in these cases than in the bron
chial cases not infected with these bacilli- (7) Result of treatment. Of the 27 cases
were treated (5 did not come for treatment and the results of treatment are only avails in 21 cases. Of these 21, 13 were completely relieved of their symptoms (there was 1 relap8^ in these 13), 3 were much improved, the m
tervals between the attacks having increased an^ the duration of the attacks lessened, while were only slightly improved and 1 not improve^ at all. (In bronchial cases without the Gram' negative-bacillus infection, of the 49 eases
treated 28 were relieved, 12 much improved, improved, and 1 not improved.) ,
The following additional points which are no included in the table are worth mentioning-
(1) The co-existence of other allergic man1' festations. In our total series of 150 cases
0
asthma there was only one case suffering f1'0^ allergic dermatitis, and it was one of thes
Gram-negative-bacillus cases.
(2) History of previous allergy. Out of tm
series only 2 gave a history of having suffere from urticaria, and two suffered from sneezmg and running of the nose before getting asthma- (In the total series of 150 cases 11 had suffereC from urticaria previously.)
(3) Dermal tests were performed with e*
tracts of the different hairs, feathers and f?? .
in general use, when indicated in the history' grass pollens were included. Of the 25 case
tested only 3 gave positive results, one ^vlt
cattle-hair, one with several foods and on
with Timothy-grass pollens and milk. "
positive tests did not prove to be of any vain from the treatment point of view. (In the bronchial cases without the Gram-negate^ bacillus infection in whom dermal tests done 57 were negative, 3 doubtful and only positive.)
(4) Yon Pirquet tests. Of the 16 cases whef this test was done only 7 were positive, y ̂
the bronchial cases without this bacillus infeC tion 35 out of 60 were positive.)
(5) Evidences of pulmonary tuberculosa1 There were only 3 cases showing any evidenc of this disease and all these were old heale^ lesions detected in the skiagrams. (In the
'
bronchial cases there were 15 tuberculous case~ of which one was an early case, 4 with i on
advanced lesions, and 10 with old lesions.) f
(6) In none of the 27 cases did a change 0
place or food have any effect on the attacks-
April, 1933] ANTI-GAMETOCYTE TREATMENT : CLEMESHA 199
Discussion.?Relation of the bacillus to
asthma.
As already mentioned Oriel (1932) considers
that the local production of histamine in the
wonchi in addition to causing a contraction of
the plain muscle surrounding the bronchi would
? 3 +V.P permeability of the the plain muscle surrounding tne "T"
also tend to increase the permeability o e
epithelium lining the bronchioles and facilitate
the entrance of foreign proteins and possibly
bacteria. Knott in a personal communication
says that
he thinks that the weight of evidence is that
a ?? ^-.rnlrp a,tl
Knott in a personal c0?"7^ence is that he thinks that the weight of e nvoice an
the organisms cannot themselves p ergon
attack of asthma in a perfectly no.rraal person, hut only in those whose ^ronc^10 +rr,0+i0n- in irritable and on the verge of
allergies these infections are t er attacks
cularly potent in precipitating clmica ?r maintaining chronic asthma.
rv j J. -fnii under the allergic Our cases do not fall unac
dermal group as is evident from the results
_
tests and from the absence of any nii 2V elated allergy or history of Prevl0+U?^2 anv We have not succeeded in demons 1 ?
ms
histamine-like effect in cultures of the org ^ isolated by us. The only thuig we can
say ^
Present is that in our stu^ ?* ^ases which
asthma we have come across 27 , +^e
constitute a clinical group among,A t
bronchial cases characterised by ,g hic eosinophilia and presence of P/le0^"lt^res Gram-negative-bacilli in smears an
?* sputum.
Summary.
1. Morphological and cultural actors^ the Gram-negative-bacilli isolated rinssi- sputa of asthmatics are described. -n; are
fieation and significance of these bacilli are
discussed. . ,i
2- A table of the cases showing n
Gram-negative-bacillus in their sputa and the following conclusions made. _
,
(a) All the cases show a how??a eosinophilia and less than one-thir sPutum eosinophilia. . f
, (b) About half the cases gave a history o
heredity. (c) Age of onset in more than three-quar
ei..
the cases varies from 11 to 30 years.
id) The response to treatment by va^1^ *nade from these organisms is the
_
same
bronchial cases due to other bacteria. (e) In only one case was there any o> lei
eo-existing allergic manifestation, but t^o 0c
a history of having suffered from urticaria ana
another two patients suffered from ?.neez!;?g and running from the nose before getting asthma.
(/) The dermal tests are of no value in the
diagnosis of these cases. (Continued at foot of next column)
(Continued from previous column)
References
Harkavy, J. (1930). Arch. Intern. Med., Vol. XLV, p. 641.
Knott, F. A. (1929). Guy's Hosp. Rep., Vol. LXXIX, p. 491. Knott, F. A., and Oriel, G. H. (1930). Journ. Physiol.,
Vol. LXX, p. xxi. Knott, F. A., Oriel, G. H., and Witts, L. J. (1930).
Guy's Hosp. Rep., Vol. LXXX, p. 421. McDowall, R. J. (1932). Lancet, Vol. I, p. 1343. Noronha, A. J. (1926). Indian Med. Gaz., Vol. LXI,
p. 75. Oriel, G. H. (1932). Allergy. London: John Bale
Sons & Danielsson, Ltd. Rackemann, F. M. (1920). Journ. Immunol., Vol V
p. 373. Rackemann, F. M., and Graham, L. B. (1923). Journ.
Immunol., Vol. VIII, p. 295. Thomas, W. S., Famulener, L. W., and Touart M D
(1924). Arch. Intern. Med., Vol. XXXIV, p. 85. Walker, C. (1919). Arch. Intern. Med., Vol XXIII
p. 220.