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THE BACTERIOLOGY OF CHRONIC BRONCHITIS
534 gave abnormal results, but all with typical persistent attacks gave results either within or very near the recommended abnormal range. There are probably individual variations in the rate of gastric emptying which will induce dumping attacks or hypoglycæmic attacks. The indications of precipitate gastric emptying likely to induce dumping attacks are : (1) an almost empty stomach at 10 minutes after a standard barium meal in the erect posture, and (2) an " absorption index " of over 600 mg. per 100 ml. on the specified oral glucose-tolerance test. (Values above or near this range may suffice to confirm a clinical suspicion of this disability, but much lower values should suffice to exclude disabling dumping attacks.) These tests may thus be useful both in selecting cases for operative reconstruction or other treatment and also in assessing the effects of such treatment. Postprandial attacks occurring more than P/2 hours after meals are usually hypoglycsemic ; they are usually associated with a low blood-sugar and are relieved by sugar. But if need be, the disability in these patients can also be assessed by an intravenous insulin-tolerance test on which it is suggested by a hypoglycaemic index " under 180 mg. per 100 ml. or near this range. The hypoglycaemic attacks are usually mild and need little direct attention ; frequent meals can avert them. As a preoperative liability to hypoglycmmia, associated with general nervousness, is probably the main basis of these attacks, additional treatment should be directed to the patient’s nervous state and to any malnutrition which would exacerbate it. During the early postoperative months, dumping attacks need little direct
symptom-free patients
our
patients
"
attention, for most are minor and do not persist. If, however, they call for attention, adequate control is
usually
achieved
by general
attention to the
THE BACTERIOLOGY OF CHRONIC BRONCHITIS J. ROBERT MAY M.D. Camb. SENIOR LECTURER IN OF THE
BACTERIOLOGY, INSTITUTE OF DISEASES CHEST, BROMPTON, LONDON
THE organisms which can be isolated from the sputum in chronic bronchitis include virtually all of those found in the normal healthy throat. Marshall (1931) sta tos that " pneumococci," streptococci, M. catarrhalis, or B. Friedldänder are to be found in the sputum in practically every case." Southwell (1946) remarks : "The organisms ... include Micrococcus catarrhalis, pneumococci, various forms of strepto. cocci, both hæmolytic and non-haemolytic, especially Strepto. coccus viridans, together with Friedlander’s bacillus and staphylococci. The organisms are almost always mixed, and it is not possible to say that any of the above predominates in the majority of cases." Mulder et al. (1952), who reported on the high incidence of Hæmophilus influenzæ in chronic bronchitis in Holland, found this organism in pure culture in the sputum of half of their cases, and concluded that it is a very important satiological factor in the disease, since its removal by chemotherapy causes an improvement in the patient’s condition. But this finding of the high incidence of pure cultures of H. influenzæ in the sputum of chronic bronchitics is at variance with the frequency of this organism reported by workers in this
country. For example, Howell (1951) found it to be the predominant organism in the sputum of only 2% of 50 cases of chronic bronchitis. The frequency with which various organisms were found to predominate in Howell’s series was as follows:
patient’s
nutrition, the giving of frequent small dry meals, high in protein and low in carbohydrate, and lying down after meals. When frequent or severe attacks have persisted for a year after the operation these measures may not suffice, and further treatment may be needed. At this stage, the next step might well be a barium meal, and an oral glucose-tolerance test performed as described above, to assess the rate of gastric emptying. If this is precipitate, operative reconstruction of the gastrectomy would then seem reasonable. Possibly a further useful preoperative test might be a therapeutic test with subcutaneous hexamethonium bromide, given half an hour before meals, and starting with 25 mg. : some relief by this means should strengthen the indications for operative reconstruction. ’
Summary
Detailed studies
made of seventeen patients 9 months after gastrectomy, to early or " dumping " (7 + 2) or to late or hypoglycaemic (8 + 2) postprandial attacks of palpitations and weakness ; and constrasted with similar studies of nine patients free of such symptoms after gastrectomy. Gastric emptying, found rapid in all gastrectomy patients, was precipitate only in the group liable to
liable,
at
were
over
dumping
attacks ;; precipitate emptying was indicated either by an empty stomach at 10 minutes after a standard barium meal, or on an oral glucose-tolerance test by a absorption index (the very rapid initial rise-the sum of the blood-sugar value at 15, 30, and 45 minutes) being over 600 mg. per 100 ml. The group liable to hypoglycæmic attacks was distinguishable on an intravenous insulin-tolerance test by an abnormal persistence of hypoglycæmia— the " hypoglycæmic index " (the sum of the bloodsugar values at 60, 90, and 120 minutes) being under 180 mg. per 100 ml. "
"
In a preliminary communication (May 1952) I showed that in many cases of chronic bronchitis some of the organisms are distributed quite irregularly throughout the sputum, so that a single culture may well give an incomplete picture of the bacteriological state of the sputum as a whole. I describe here details of the technique used in these investigations, together with the
bacteriological findings. Materials and Methods
Collection of Sputum and its Preparation for Culture A specimen of sputum, as used in this investigation, is defined as a portion of sputum expectorated as a result of one bout of coughing only. Each specimen examined was expectorated by the patient into a sterile screwuniversal container," to which was added capped subsequently about five times as much sterile physiological saline solution. The sputum was well rinsed in the saline solution before being transferred to an empty sterile petri dish. In this way much of the adherent saliva "
"
"
Adlersberg, D., Hammerschlag, K. (1947) Surgery, 21, 720. (1949) J. Amer. med. Ass. 139, 429. Alvarez, W. C. (1939) Gastroenterol. 13, 212. Bornstein, J., Trewhella, P. (1950) Aust. J. exp. Biel. med. Sci. 28, 569. Conn, J. W. (1940) J. Amer. med. Ass. 115, 1669. Edlen, A. (1950) Acta med. scand. 138, 161. Evensen, O. K. (1942) Ibid, suppl. 126. Fraser R. (1943) M.D. thesis, University of Otago, N.Z. Unpublished. Smith, P. (1941) Quart. J. Med. 10, 297. Glazebrook, A. J., Welbourn, R. B. (1952) Brit. J. Surg. 40, 111. Hertz, A. F. (1913) Ann. Surg. 58, 466. Hildes, S.A., Sherlock, S., Walshe, V. (1949) Clin. Sci. 7, 297. Kendall, E. C. (1938) Proc. Mayo Clin. 13, 519. Moore, A. (1950) Med. J. Aust. 1, 563. Smith, W. H. (1951) Lancet, ii, 745. Fraser, R., Staynes, K., Willcox, J. M. (1953) Quart. J. Med. (in the press). Wilder, R. M. (1940). Clinical Diabetes Mellitus and Hyperinsulinism. Philadelphia and London. —
—
—
—
535 removed. In no instance did more than six hours elapse between the expectoration of the sputum and the taking of cultures. In most cases this interval was considerably shorter. was
Ratadom The ruled
SampUng
petri onf)
dish
of Taking Cultures containing the sputum was Method
nf numbered
squares,
each
laid
about
on
a
0.75
x:
0-75 cm. (fig.
J).
From a. table off random numbers (Fisher and Yates 1948) the first five
numbers were selected whicli
corresponded
to
numbers on the grid covered by the sputum. These indicated the sites from which cultures e to be were made.** In each instance a note was made as to whether the
to illustrate use of random numbers for selection of sites from which cultures should be made from a specimen
Fig. I-Diagram of sputum.
culture
was predominantly mucoid or purulent. making a culture care was taken to ensure that the wire loop was not simply dipped into the sputum and withdrawn with only a little moisture adhering to it. Such moisture might well consist mainly of surface saliva
and the relative numbers of the colonies of each bacterial species present were calculated. These were expressed as percent.ages of the total number counted. For Much counting to be accurate the sputum had to be broken ulr very thoroughly before being spread, as mentioned earlier. To facilitate the counting, an electric counter similar’ to that devised by Linsell (1953) was UMed.
microscope,
Throat Swabs Swabs of the throat
examined for many of the Counts were not jnn.de on these culture.s. the relative numbers of each type of colony were
patients investigated.
being assessed by simple inspection only. Culture Media
Blood-agar was used as the standard medium on which primary cultures were made. Any organisms not easily recognised on blood-agar were irlentified by subculture on to other suitable media before the appropriate diagnostic tests -were made.
all the
Subjects Investigated ’I’he patients oil whom
these
investigations
were
made
fell into two groups :
(1)
-)4
the bronchitis clinic at the From each of these patients only one
outpatients a,ttending
Brompton Huspita1.
TABLE I—DISTRIBUTION
In
OF
SPUTUM
No. of
which had not been removed by the preliminary washing. and the organisms contained in it would not necessarily reflect the true flora in that part of the sputum. The loop was used to extract a small portion of actual sputum from the site selected. This piece of sputum was then transferred to the side of the culture medium, where it was very thoroughly broken up with the loop before heing spread over the plate. In this way not only was the risk of salivarv contamination minimised but also the bacterial eolonies were evenly distributed, with relative
No. of Five cultures
No.
I times isolated
Orgamism
SPECIMENS
ORGANISMS IN SINGLE
OF
of
times
distributed ! evenly
No. of times
distributed unevenly
specimens examined = 54.
made from each specimen of sputum. If an organism was present in all 5 cultures it was classed as evenly distributed." Organisms present in 1, 2, 3, or 4 cultures only were classed as unevenly distributed." were
w
"
specimen of sputum was obtained and examined bacteriologically by the five-culture method described above to obtain information about the distribution of organisms within single specimens of sputum. Throat swabs also were taken from many of these patients. (2) 14 inpatients in the Brompton Hospital. The variations
Fig. 3-Distribution of
of organisms in different parts of a single specimen of sputum. Diagram shows sites from which cultures listed in table were made.
Fig. 2-Distribution
organdifferent parts of a single specimen of sputum. Diagram shows sites from which cultures listed in table were made. isms
freedom from the " strea.kine!ss cultures of sputum.
"
so
in
commonly
seem
ill.
in the bacterial content of the
sputum from specimen to specimen and from day to day were investigated in these patients. In each case the procedure was the same. Three specimens of sputum, produced consecutively, were examined on one day ; one specimen was examined the next day, and one the following day. Finally, a single specimen was examined Five cultures a week after the first specimens were obtained. followed by differential colony counting, were made from each specimen. Results
Differential Colony Counting Each of the five cultures made from a specimen of was subjected to differential colony counting." In this procedure as iiiiiiy colonies as possible on each plate were counted with the aid of a binocular dissecting
sputum
OF BACTERIA WITHIN SPECIMENS OF SPUTUM
DISTRIBUTION
"
*In early experiinents cultures
were it from thirty HiteH in each sputum specimen, and differential colony counts were made on each culture. The estimate of the proportions of the various bacterial species in tho specimen obtained by usinK all thirty sites was compared with the estimates obtained by using random samples of various sixes. Estimates based on five sites differed only slightly from those based on thirty sites, whereas the results obtained from smaller samples often varied widely. Cultures from five parts of each specimen were therefore concluded to be Hufficient for praeticat purposes.
Fig.
SINGLE
2 shows the relative numbers of different organ-
isms
present in various parts of a single specimen of sputum. This sputum shows extrerne variations of flora from one part to another. Though the proportion of each organism present shows some variation in different cultures, the numbers of Strep. viridans, non-hæmolytic
streptococci, and N. catarrhaZis remain substantially constant in relation to each other in all the cultures. The proportion of pneumococci, however, is clearly not
536 TABLE
II—DISTRIBUTION
SPUTUM OF WHICH
27
OF
H.
influenzæ
DIFFERENT
SITES
IN
SPECIMEN
A
WERE
OF
CULTURED
related to that of any of these organisms, suggesting that the pneumococcus is the only organism wliose distribution is irregular in this sputum. A similar state of a,ffairs is apparent in the sputum illustrated by hg. 3. In this instance II. influenzæ seems to be the variable organism, while Strep. viridans, nom-li.rmolvtic streptococci, and N. catarrhat-is are again substantially constant. The pneumococcus and H. influenzæ, both of which are known to be potential pathogens in the respiratory tract, are the two organisms which have shown the most dramatic variations in the sputa examined in this investigation. Organisms whose pathogenic propensities are known to be slight or non-existent—e.g., Strep. viridans, non-hæmolytic streptococci, and N.catarrhalis - seem to be distributed most frequently in a more or less regular manner throughout the sputum. On occasion, however, pneumococci and H. influenzæ are (list i-i 1)1-ited evenly throughout a sputum specimen, just as one may find, though rarely, a sputum in which a shows considerable variations. The frequency with which different organisms have been found to be distributed evenly and unevenly throughout 54 specimens of sputum is shown in table i. An example of the irregular distrihution of Staph. aureus within a specimen of sputum is shown in fig. 4.
Examination of sputum by a multiple-culture tech. nique his 41iowii that pathogens " are not always isolated more frequently from purulent parts of a specimen than from mucoid parts. Cultures were made from four purulent; sites in the sputum illustrated in fig. 2 yet pneumonococci were. present in only one of these. Similarly, IT. influenzæ was present in only two out of four purulent sites in the sputum shown in fig. 3. Table n shows the distribution of II. influenzæ in a specimen of sputum from which 27 cultures were made. Clearly the chance of isolating this organism by means of a single culture was no greater if the site cultured was purulent than if it was mucoid. Though the distribution of pathogens " in a specimen of sputum does not necessarily correspond to that of the pus, purulent sputa are more likely to contain these organisms than are mucoid sputa. Organisms of doubtful pathogenicity, however, seem to be found with equal frequency in both types of sputum. This is shown ill "
"
TABLE III—INCIDENCE OF VARIOUS ORGANISMS IN MUCOID AND PURULENT SPUTA
non-pathogen
Relation
of Organisms to Pus in Sputum organisms in sputum can be considered conveniently in two groups : The
most
(1) Those which are known to be potent ial respiratory pathogens : the pneumococcus. H. influenzæ, Staph. aureus and Friecllander’s bacillus. These organisms will be referred to hereafter as pathogens." (2) Those of doubtful pat.hobenieity : Strep. viridans, nonliapmolytic streptococei, N. catarrhalis, diphtheroid bacilli, Staph. czlbzes, and ooliform bacilli. "
Sputum
specimens were classified as "mucoid" or "purulent" mainly- I)N- maked-eye inspection. In cases of doubt microscopy was also used. puta consisting of mucus containing lumps of pus were classed as purulent." All thesputa were examined by the five"
culture method.
Full details of the distribution of the group of in mucoid and purulent sputa -are given in table iv. It will be seen that a probable pathogen was demonstrable in 90% of purulent sputa, whereas in mucoid sputa the corresponding figure was only 54-2,’ . This difference is most unlikely to be due to chance. The results shown in table iv were obtained from the study of single specimens of sputum from each patient. Of the 3 patients with purulent sputa in which no were " pathogen " was demonstrable, further specimens examined from 2. Each of these revealed a " pathogen one 11. influenzæ, the other a pneumococcus.
table "
III.
pathogens
"
VARIATIONS OF BACTERIAL FLORA IN DIFFERENT SPECIMENS OF SPUTUM FROM SAME PATIENT
Investigations have
been made on 14 patients to deterof cultures from a single specimen of sputum as an index of the flora of the whole bronchial tree. Reference has already been made to 2 patients from whom more than one specimen of sputum was required to reveal a " pathogen." Each of the 14 patients mine the
reliability
producing consistently purulent or mucopurulent 11 cases a pathogen " was revealed in the first specimen examined ; in 2 cases " pathogens " were found in the next specimen expectorated ; and in the remaining case a pathogen (pneumococcus) was found only in the sixth specimen examined-i.e., a week after the production of the first sample. The pathogens" isolated from all of these sputa were either H. influenzæ or pneumococci, or both organisms together. It is of some interest that in one patient the first specimen of sputum contained pneumococci, whereas another speci. was
"
sputum. In
"
"
"
Fig. 4-Example irregular distribution of Staph. aureus in a single specimen of sputum. These four cultures were made from a specimen which covered an area about 1.5 : 1.5 cm. in a petri dish. The The large creamy sputum consisted of homogeneous mucopus. colonies are those of Stabh. aureus. of
537 men,
expectorated only 10 minutes later, influenzæ also.
contained
large
numbers of H.
from which only one culture would be required to reveal a,lt tin’ species of bacteria present. Such a method has now been developed by Rawlins (L!)53). The irregular distribution of some organisms in sputum provides a possible C’xplanatioll for the variations observed by Howell (1951) in weekly cultures from the sputum of chronic bronchitics. It does not provide a complete explanation, however, because in some patients differences were observed in the flora of different specimens of sputum, even when examined by the five-culture More than one specimen from any patient, method. therefore, should be examined before it is concluded that all the species of bacteria have been revealed. This is especially important in patients with a purulent sputum from which no obvious pathogens can be isolated at first. In the present series organisms known to be potential pathogens could always be demonstrated in such patients, provided that enough samples of sputum .
BACTERIAL FLORA OF SPUTUM TN RELATION TO THAT OF THROAT SWABS
of
throat swabs were made from 28 patients, whose sputum was also examined. 1’otemtially pathogenic organisms were found in the sputum of 23 patients ; but the same organisms could be isolated from the throats of only 12 of these. In 4 other patients, in all of whom two "pathogens" were present in the sputum, one "pathogen" only was "found in the throat. In 1 other (pneumococcus) was isolated "’pathogen from the throat but was not found in the sputum, which was mucoid. In contrast to the findings for pathogenic organisms, the same non-pathogens were present in both the sputum and throat swab of each of the 28 patients investigated.
Cultures
patient a
were
Discussion
The method of making a culture from sputum in use in most bacteriological laboratories involves only a single "loopful"of the material. The results presented here indicate that such a procedure may fail to reveal all the species of bacteria present in the whole specimen, and the precaution of making a culture from a purulent part of the sputum does not necessarily enhance the reliahility of the technique. In the investigations described here cultures were made from five different sites of each specimen of sputum examined. Such a technique is clearly not practicable in a routine laboratory, and a method is required for liquefying the sputum without interfering-with the viability of the organisms it contains. In this way a homogeneous suspension could be prepared TABLE IV-INCIDENCE OF POTENTIAL. PATHOGENS AND PURULENT SPUTA
IN
MUCOID
examined.
to he potential pathogens in the respiratory tract, especially the pneumococcus and II. influenzæ, have been shown to he significantly related to the presence of pus in the sputum, Proof that these organisms are pathogens in chronic bronchitis depends on 1 he demonstration that their removal from the sputum by chemotherapy, all other organisms remaining unchanged, is associated with improvement in the patient’s condition. Preliminary studies on these lines,
Organisms known
reported
details of which will be later, have shown that the removal of pneumococci or of H. influenzæ from the respiratory tract of any patient with a purulent sputum has been associated in each case with diminution, and often elimination, of the pus. The response to antibiotics is also being used as a means of investigating the significance of potential pathogens in mucoid sputa (see table iv). The eradication of these organisms does not seem to lead to diminution of the quantity of a purely
mucoid
sputum. Summary
A single culture from a specimen of sputum often fails to reveal all the species of bacteria present, even though the culture is made from a purulent part. Cultures should be made from at least five different parts of each specimen to obtain a reliable estimate of the proportions of each organism present. The need for a method of homogenising the sputum, without interfering with bacterial viability, is indicated. Single specimens of sputum may fail to reveal all the species of bacteria present in the bronchial tree of a chronic bronchitic. At least two, and preferably more, samples should be examined. There is a significant association between pus in the sputum and the presence of potential pathogens, especially the pneumococcus and H. influenzæ. Organisms of doubtful pathogenicity, such as Strep. viridans, nonhæmolytic streptococci, and N. catarrhalis, are not related to pus in the sputum. I wish to express my thanks to Dr. Neville Oswald for his and for permission to investigate his patients ; Mr. G. A. Rawlins for his technical assistance ; Dr. D. D. Reid for advice on statistical matters ; and Mr. D. F. Kemp for the photograph.
cooperation
REFERENCES
+=Organisms present but not in suniciont quantity to be shown in differential count.
Fisher, R. A., Yates, F. (1948) Statistical Tables for Biological, 3rd ed., Edinburgh ; Agricultural, and Medical Research. p. 104. Howell, T. H. (1951) Chronic Bronchitis. London ; p. 60. Linsell, W. D. (1953) J. clin. Path. 6, 91. Marshall, G. (1931) Practitioner, 126, 59. May, J. R. (1952) Lancet, ii, 1206. Mulder, J., Goslings, W. R. O., van der Plas, M. C., Lopes Cardozo, P. (1952) Acta med. scand. 143, 32. Rawlins, G. A. (1953) Lancet, ii, 538. Southwell, N. (1946) Brit. J. industr. Med. 3, 75.
symptom-free patients
our
patients
"
attention, for most are minor and do not persist. If, however, they call for attention, adequate control is
usually
achieved
by general
attention to the
THE BACTERIOLOGY OF CHRONIC BRONCHITIS J. ROBERT MAY M.D. Camb. SENIOR LECTURER IN OF THE
BACTERIOLOGY, INSTITUTE OF DISEASES CHEST, BROMPTON, LONDON
THE organisms which can be isolated from the sputum in chronic bronchitis include virtually all of those found in the normal healthy throat. Marshall (1931) sta tos that " pneumococci," streptococci, M. catarrhalis, or B. Friedldänder are to be found in the sputum in practically every case." Southwell (1946) remarks : "The organisms ... include Micrococcus catarrhalis, pneumococci, various forms of strepto. cocci, both hæmolytic and non-haemolytic, especially Strepto. coccus viridans, together with Friedlander’s bacillus and staphylococci. The organisms are almost always mixed, and it is not possible to say that any of the above predominates in the majority of cases." Mulder et al. (1952), who reported on the high incidence of Hæmophilus influenzæ in chronic bronchitis in Holland, found this organism in pure culture in the sputum of half of their cases, and concluded that it is a very important satiological factor in the disease, since its removal by chemotherapy causes an improvement in the patient’s condition. But this finding of the high incidence of pure cultures of H. influenzæ in the sputum of chronic bronchitics is at variance with the frequency of this organism reported by workers in this
country. For example, Howell (1951) found it to be the predominant organism in the sputum of only 2% of 50 cases of chronic bronchitis. The frequency with which various organisms were found to predominate in Howell’s series was as follows:
patient’s
nutrition, the giving of frequent small dry meals, high in protein and low in carbohydrate, and lying down after meals. When frequent or severe attacks have persisted for a year after the operation these measures may not suffice, and further treatment may be needed. At this stage, the next step might well be a barium meal, and an oral glucose-tolerance test performed as described above, to assess the rate of gastric emptying. If this is precipitate, operative reconstruction of the gastrectomy would then seem reasonable. Possibly a further useful preoperative test might be a therapeutic test with subcutaneous hexamethonium bromide, given half an hour before meals, and starting with 25 mg. : some relief by this means should strengthen the indications for operative reconstruction. ’
Summary
Detailed studies
made of seventeen patients 9 months after gastrectomy, to early or " dumping " (7 + 2) or to late or hypoglycaemic (8 + 2) postprandial attacks of palpitations and weakness ; and constrasted with similar studies of nine patients free of such symptoms after gastrectomy. Gastric emptying, found rapid in all gastrectomy patients, was precipitate only in the group liable to
liable,
at
were
over
dumping
attacks ;; precipitate emptying was indicated either by an empty stomach at 10 minutes after a standard barium meal, or on an oral glucose-tolerance test by a absorption index (the very rapid initial rise-the sum of the blood-sugar value at 15, 30, and 45 minutes) being over 600 mg. per 100 ml. The group liable to hypoglycæmic attacks was distinguishable on an intravenous insulin-tolerance test by an abnormal persistence of hypoglycæmia— the " hypoglycæmic index " (the sum of the bloodsugar values at 60, 90, and 120 minutes) being under 180 mg. per 100 ml. "
"
In a preliminary communication (May 1952) I showed that in many cases of chronic bronchitis some of the organisms are distributed quite irregularly throughout the sputum, so that a single culture may well give an incomplete picture of the bacteriological state of the sputum as a whole. I describe here details of the technique used in these investigations, together with the
bacteriological findings. Materials and Methods
Collection of Sputum and its Preparation for Culture A specimen of sputum, as used in this investigation, is defined as a portion of sputum expectorated as a result of one bout of coughing only. Each specimen examined was expectorated by the patient into a sterile screwuniversal container," to which was added capped subsequently about five times as much sterile physiological saline solution. The sputum was well rinsed in the saline solution before being transferred to an empty sterile petri dish. In this way much of the adherent saliva "
"
"
Adlersberg, D., Hammerschlag, K. (1947) Surgery, 21, 720. (1949) J. Amer. med. Ass. 139, 429. Alvarez, W. C. (1939) Gastroenterol. 13, 212. Bornstein, J., Trewhella, P. (1950) Aust. J. exp. Biel. med. Sci. 28, 569. Conn, J. W. (1940) J. Amer. med. Ass. 115, 1669. Edlen, A. (1950) Acta med. scand. 138, 161. Evensen, O. K. (1942) Ibid, suppl. 126. Fraser R. (1943) M.D. thesis, University of Otago, N.Z. Unpublished. Smith, P. (1941) Quart. J. Med. 10, 297. Glazebrook, A. J., Welbourn, R. B. (1952) Brit. J. Surg. 40, 111. Hertz, A. F. (1913) Ann. Surg. 58, 466. Hildes, S.A., Sherlock, S., Walshe, V. (1949) Clin. Sci. 7, 297. Kendall, E. C. (1938) Proc. Mayo Clin. 13, 519. Moore, A. (1950) Med. J. Aust. 1, 563. Smith, W. H. (1951) Lancet, ii, 745. Fraser, R., Staynes, K., Willcox, J. M. (1953) Quart. J. Med. (in the press). Wilder, R. M. (1940). Clinical Diabetes Mellitus and Hyperinsulinism. Philadelphia and London. —
—
—
—
535 removed. In no instance did more than six hours elapse between the expectoration of the sputum and the taking of cultures. In most cases this interval was considerably shorter. was
Ratadom The ruled
SampUng
petri onf)
dish
of Taking Cultures containing the sputum was Method
nf numbered
squares,
each
laid
about
on
a
0.75
x:
0-75 cm. (fig.
J).
From a. table off random numbers (Fisher and Yates 1948) the first five
numbers were selected whicli
corresponded
to
numbers on the grid covered by the sputum. These indicated the sites from which cultures e to be were made.** In each instance a note was made as to whether the
to illustrate use of random numbers for selection of sites from which cultures should be made from a specimen
Fig. I-Diagram of sputum.
culture
was predominantly mucoid or purulent. making a culture care was taken to ensure that the wire loop was not simply dipped into the sputum and withdrawn with only a little moisture adhering to it. Such moisture might well consist mainly of surface saliva
and the relative numbers of the colonies of each bacterial species present were calculated. These were expressed as percent.ages of the total number counted. For Much counting to be accurate the sputum had to be broken ulr very thoroughly before being spread, as mentioned earlier. To facilitate the counting, an electric counter similar’ to that devised by Linsell (1953) was UMed.
microscope,
Throat Swabs Swabs of the throat
examined for many of the Counts were not jnn.de on these culture.s. the relative numbers of each type of colony were
patients investigated.
being assessed by simple inspection only. Culture Media
Blood-agar was used as the standard medium on which primary cultures were made. Any organisms not easily recognised on blood-agar were irlentified by subculture on to other suitable media before the appropriate diagnostic tests -were made.
all the
Subjects Investigated ’I’he patients oil whom
these
investigations
were
made
fell into two groups :
(1)
-)4
the bronchitis clinic at the From each of these patients only one
outpatients a,ttending
Brompton Huspita1.
TABLE I—DISTRIBUTION
In
OF
SPUTUM
No. of
which had not been removed by the preliminary washing. and the organisms contained in it would not necessarily reflect the true flora in that part of the sputum. The loop was used to extract a small portion of actual sputum from the site selected. This piece of sputum was then transferred to the side of the culture medium, where it was very thoroughly broken up with the loop before heing spread over the plate. In this way not only was the risk of salivarv contamination minimised but also the bacterial eolonies were evenly distributed, with relative
No. of Five cultures
No.
I times isolated
Orgamism
SPECIMENS
ORGANISMS IN SINGLE
OF
of
times
distributed ! evenly
No. of times
distributed unevenly
specimens examined = 54.
made from each specimen of sputum. If an organism was present in all 5 cultures it was classed as evenly distributed." Organisms present in 1, 2, 3, or 4 cultures only were classed as unevenly distributed." were
w
"
specimen of sputum was obtained and examined bacteriologically by the five-culture method described above to obtain information about the distribution of organisms within single specimens of sputum. Throat swabs also were taken from many of these patients. (2) 14 inpatients in the Brompton Hospital. The variations
Fig. 3-Distribution of
of organisms in different parts of a single specimen of sputum. Diagram shows sites from which cultures listed in table were made.
Fig. 2-Distribution
organdifferent parts of a single specimen of sputum. Diagram shows sites from which cultures listed in table were made. isms
freedom from the " strea.kine!ss cultures of sputum.
"
so
in
commonly
seem
ill.
in the bacterial content of the
sputum from specimen to specimen and from day to day were investigated in these patients. In each case the procedure was the same. Three specimens of sputum, produced consecutively, were examined on one day ; one specimen was examined the next day, and one the following day. Finally, a single specimen was examined Five cultures a week after the first specimens were obtained. followed by differential colony counting, were made from each specimen. Results
Differential Colony Counting Each of the five cultures made from a specimen of was subjected to differential colony counting." In this procedure as iiiiiiy colonies as possible on each plate were counted with the aid of a binocular dissecting
sputum
OF BACTERIA WITHIN SPECIMENS OF SPUTUM
DISTRIBUTION
"
*In early experiinents cultures
were it from thirty HiteH in each sputum specimen, and differential colony counts were made on each culture. The estimate of the proportions of the various bacterial species in tho specimen obtained by usinK all thirty sites was compared with the estimates obtained by using random samples of various sixes. Estimates based on five sites differed only slightly from those based on thirty sites, whereas the results obtained from smaller samples often varied widely. Cultures from five parts of each specimen were therefore concluded to be Hufficient for praeticat purposes.
Fig.
SINGLE
2 shows the relative numbers of different organ-
isms
present in various parts of a single specimen of sputum. This sputum shows extrerne variations of flora from one part to another. Though the proportion of each organism present shows some variation in different cultures, the numbers of Strep. viridans, non-hæmolytic
streptococci, and N. catarrhaZis remain substantially constant in relation to each other in all the cultures. The proportion of pneumococci, however, is clearly not
536 TABLE
II—DISTRIBUTION
SPUTUM OF WHICH
27
OF
H.
influenzæ
DIFFERENT
SITES
IN
SPECIMEN
A
WERE
OF
CULTURED
related to that of any of these organisms, suggesting that the pneumococcus is the only organism wliose distribution is irregular in this sputum. A similar state of a,ffairs is apparent in the sputum illustrated by hg. 3. In this instance II. influenzæ seems to be the variable organism, while Strep. viridans, nom-li.rmolvtic streptococci, and N. catarrhat-is are again substantially constant. The pneumococcus and H. influenzæ, both of which are known to be potential pathogens in the respiratory tract, are the two organisms which have shown the most dramatic variations in the sputa examined in this investigation. Organisms whose pathogenic propensities are known to be slight or non-existent—e.g., Strep. viridans, non-hæmolytic streptococci, and N.catarrhalis - seem to be distributed most frequently in a more or less regular manner throughout the sputum. On occasion, however, pneumococci and H. influenzæ are (list i-i 1)1-ited evenly throughout a sputum specimen, just as one may find, though rarely, a sputum in which a shows considerable variations. The frequency with which different organisms have been found to be distributed evenly and unevenly throughout 54 specimens of sputum is shown in table i. An example of the irregular distrihution of Staph. aureus within a specimen of sputum is shown in fig. 4.
Examination of sputum by a multiple-culture tech. nique his 41iowii that pathogens " are not always isolated more frequently from purulent parts of a specimen than from mucoid parts. Cultures were made from four purulent; sites in the sputum illustrated in fig. 2 yet pneumonococci were. present in only one of these. Similarly, IT. influenzæ was present in only two out of four purulent sites in the sputum shown in fig. 3. Table n shows the distribution of II. influenzæ in a specimen of sputum from which 27 cultures were made. Clearly the chance of isolating this organism by means of a single culture was no greater if the site cultured was purulent than if it was mucoid. Though the distribution of pathogens " in a specimen of sputum does not necessarily correspond to that of the pus, purulent sputa are more likely to contain these organisms than are mucoid sputa. Organisms of doubtful pathogenicity, however, seem to be found with equal frequency in both types of sputum. This is shown ill "
"
TABLE III—INCIDENCE OF VARIOUS ORGANISMS IN MUCOID AND PURULENT SPUTA
non-pathogen
Relation
of Organisms to Pus in Sputum organisms in sputum can be considered conveniently in two groups : The
most
(1) Those which are known to be potent ial respiratory pathogens : the pneumococcus. H. influenzæ, Staph. aureus and Friecllander’s bacillus. These organisms will be referred to hereafter as pathogens." (2) Those of doubtful pat.hobenieity : Strep. viridans, nonliapmolytic streptococei, N. catarrhalis, diphtheroid bacilli, Staph. czlbzes, and ooliform bacilli. "
Sputum
specimens were classified as "mucoid" or "purulent" mainly- I)N- maked-eye inspection. In cases of doubt microscopy was also used. puta consisting of mucus containing lumps of pus were classed as purulent." All thesputa were examined by the five"
culture method.
Full details of the distribution of the group of in mucoid and purulent sputa -are given in table iv. It will be seen that a probable pathogen was demonstrable in 90% of purulent sputa, whereas in mucoid sputa the corresponding figure was only 54-2,’ . This difference is most unlikely to be due to chance. The results shown in table iv were obtained from the study of single specimens of sputum from each patient. Of the 3 patients with purulent sputa in which no were " pathogen " was demonstrable, further specimens examined from 2. Each of these revealed a " pathogen one 11. influenzæ, the other a pneumococcus.
table "
III.
pathogens
"
VARIATIONS OF BACTERIAL FLORA IN DIFFERENT SPECIMENS OF SPUTUM FROM SAME PATIENT
Investigations have
been made on 14 patients to deterof cultures from a single specimen of sputum as an index of the flora of the whole bronchial tree. Reference has already been made to 2 patients from whom more than one specimen of sputum was required to reveal a " pathogen." Each of the 14 patients mine the
reliability
producing consistently purulent or mucopurulent 11 cases a pathogen " was revealed in the first specimen examined ; in 2 cases " pathogens " were found in the next specimen expectorated ; and in the remaining case a pathogen (pneumococcus) was found only in the sixth specimen examined-i.e., a week after the production of the first sample. The pathogens" isolated from all of these sputa were either H. influenzæ or pneumococci, or both organisms together. It is of some interest that in one patient the first specimen of sputum contained pneumococci, whereas another speci. was
"
sputum. In
"
"
"
Fig. 4-Example irregular distribution of Staph. aureus in a single specimen of sputum. These four cultures were made from a specimen which covered an area about 1.5 : 1.5 cm. in a petri dish. The The large creamy sputum consisted of homogeneous mucopus. colonies are those of Stabh. aureus. of
537 men,
expectorated only 10 minutes later, influenzæ also.
contained
large
numbers of H.
from which only one culture would be required to reveal a,lt tin’ species of bacteria present. Such a method has now been developed by Rawlins (L!)53). The irregular distribution of some organisms in sputum provides a possible C’xplanatioll for the variations observed by Howell (1951) in weekly cultures from the sputum of chronic bronchitics. It does not provide a complete explanation, however, because in some patients differences were observed in the flora of different specimens of sputum, even when examined by the five-culture More than one specimen from any patient, method. therefore, should be examined before it is concluded that all the species of bacteria have been revealed. This is especially important in patients with a purulent sputum from which no obvious pathogens can be isolated at first. In the present series organisms known to be potential pathogens could always be demonstrated in such patients, provided that enough samples of sputum .
BACTERIAL FLORA OF SPUTUM TN RELATION TO THAT OF THROAT SWABS
of
throat swabs were made from 28 patients, whose sputum was also examined. 1’otemtially pathogenic organisms were found in the sputum of 23 patients ; but the same organisms could be isolated from the throats of only 12 of these. In 4 other patients, in all of whom two "pathogens" were present in the sputum, one "pathogen" only was "found in the throat. In 1 other (pneumococcus) was isolated "’pathogen from the throat but was not found in the sputum, which was mucoid. In contrast to the findings for pathogenic organisms, the same non-pathogens were present in both the sputum and throat swab of each of the 28 patients investigated.
Cultures
patient a
were
Discussion
The method of making a culture from sputum in use in most bacteriological laboratories involves only a single "loopful"of the material. The results presented here indicate that such a procedure may fail to reveal all the species of bacteria present in the whole specimen, and the precaution of making a culture from a purulent part of the sputum does not necessarily enhance the reliahility of the technique. In the investigations described here cultures were made from five different sites of each specimen of sputum examined. Such a technique is clearly not practicable in a routine laboratory, and a method is required for liquefying the sputum without interfering-with the viability of the organisms it contains. In this way a homogeneous suspension could be prepared TABLE IV-INCIDENCE OF POTENTIAL. PATHOGENS AND PURULENT SPUTA
IN
MUCOID
examined.
to he potential pathogens in the respiratory tract, especially the pneumococcus and II. influenzæ, have been shown to he significantly related to the presence of pus in the sputum, Proof that these organisms are pathogens in chronic bronchitis depends on 1 he demonstration that their removal from the sputum by chemotherapy, all other organisms remaining unchanged, is associated with improvement in the patient’s condition. Preliminary studies on these lines,
Organisms known
reported
details of which will be later, have shown that the removal of pneumococci or of H. influenzæ from the respiratory tract of any patient with a purulent sputum has been associated in each case with diminution, and often elimination, of the pus. The response to antibiotics is also being used as a means of investigating the significance of potential pathogens in mucoid sputa (see table iv). The eradication of these organisms does not seem to lead to diminution of the quantity of a purely
mucoid
sputum. Summary
A single culture from a specimen of sputum often fails to reveal all the species of bacteria present, even though the culture is made from a purulent part. Cultures should be made from at least five different parts of each specimen to obtain a reliable estimate of the proportions of each organism present. The need for a method of homogenising the sputum, without interfering with bacterial viability, is indicated. Single specimens of sputum may fail to reveal all the species of bacteria present in the bronchial tree of a chronic bronchitic. At least two, and preferably more, samples should be examined. There is a significant association between pus in the sputum and the presence of potential pathogens, especially the pneumococcus and H. influenzæ. Organisms of doubtful pathogenicity, such as Strep. viridans, nonhæmolytic streptococci, and N. catarrhalis, are not related to pus in the sputum. I wish to express my thanks to Dr. Neville Oswald for his and for permission to investigate his patients ; Mr. G. A. Rawlins for his technical assistance ; Dr. D. D. Reid for advice on statistical matters ; and Mr. D. F. Kemp for the photograph.
cooperation
REFERENCES
+=Organisms present but not in suniciont quantity to be shown in differential count.
Fisher, R. A., Yates, F. (1948) Statistical Tables for Biological, 3rd ed., Edinburgh ; Agricultural, and Medical Research. p. 104. Howell, T. H. (1951) Chronic Bronchitis. London ; p. 60. Linsell, W. D. (1953) J. clin. Path. 6, 91. Marshall, G. (1931) Practitioner, 126, 59. May, J. R. (1952) Lancet, ii, 1206. Mulder, J., Goslings, W. R. O., van der Plas, M. C., Lopes Cardozo, P. (1952) Acta med. scand. 143, 32. Rawlins, G. A. (1953) Lancet, ii, 538. Southwell, N. (1946) Brit. J. industr. Med. 3, 75.