Quantitation of Microorganisms in Sputum

Transcription

1 APPTED MICROBIOLOGY, Aug. 1969, p VoL 18, No. 2 Copyright 1969 American Society for Microbiology Printed in U.S.A. Quantitation of Microorganisms in Sputum P. W. MONROE, H. G. MUCHMORE, F. G. FELTON, AND J. K. PIRTLE Departments ofmedicine and Microbiology, The University ofoklahoma Medical Center and Veterans Administration Hospital, Oklahoma City, Oklahoma 7314 Received for publication 25 April 1969 A method of quantitating microbial cultures of homogenized sputum has been devised. Possible application of this method to the problem of determining the etiologic agent of lower-respiratory-tract infections has been studied to determine its usefulness as a guide in the management of these infections. Specimens were liquefied by using an equal volume of 2% N-acetyl-L-cysteine. The liquefied sputum suspension was serially diluted to 1-1, 1-3, 1-5, and 1-7. These dilutions were plated on appropriate media byusing ano.1-ml calibrated loop; they were incubated and examined by standard diagnostic methods. Quantitation of fresh sputum from patients with pneumonia prior to antimicrobial therapy revealed that probable pathogens were present in populations of 17 organisms/ml or greater. Normal oropharyngeal flora did not occur in these numbers before therapy. Comparison of microbial counts on fresh and aged sputum showed that it is necessary to use only fresh specimens, since multiplication or death alters both quantitative and qualitative findings. Proper collection and quantitative culturing of homogenized sputum provided information more directly applicable to patient management than did qualitative routine methods. Not only was the recognition of the probable pathogenic organism in pneumonia patients improved, but serial quantitative cultures were particularly useful in recognizing the emergence of superinfections and in evaluating the efficacy of antimicrobial therapy. Routine qualitative microbial cultures of sputum are widely utilized in medicine to determine the etiology of lower-respiratory-tract infections and as a guide in the administration of appropriate antimicrobial agents. However, these cultures often fail to give adequate information, because the contamination of the specimen by oral flora (3, 7, 13) and the irregular distribution of organisms within the sample (8) make correct recognition of the true pathogen difficult. In such cases, quantitative microbial analyses of homogenized sputum would be helpful if they could provide better pathogen recognition. Studies of other infectious diseases, e.g., urinary tract infections, have demonstrated that determinations of the numbers of organisms present may be of considerable value in deciding which of these is causing disease and which is present as incidental colonization or recent contamination during specimen collection (4, 1, 12). Quantitation of organisms in sputum presents more problems than in urine specimens, because the former normally contains many bacteria which, of necessity, will be enumerated and because the sputum contains several substances, such as mucus, which must be liquefied before thorough mixing can be achieved. This study 214 was undertaken to develop a reliable method for obtaining a true estimate of the numbers of the various species of microorganisms present in sputum, and, if possible, to relate the numbers of resident microbial populations with respect to their significance as agents of lower-respiratorytract infections. MATERIALS AND METHODS Clinical specimens. Specimens used in the study were of three types. (i) Sputum specimens collected and submitted to the clinical microbiology laboratory of the Veterans Administration Hospital, Oklahoma City, were used. The time between specimen collection and its culture in the laboratory was approximately 4 hr, more or less. These specimens are referred to as aged sputum. (ii) Fresh sputum specimens were collected from 19 patients with clinical pneumonia who had received no antibiotics. These patients were selected by resident physicians assigned to the Infectious Disease section of the Department of Medicine, University of Oklahoma School of Medicine, Oklahoma City. Specimens were collected prior to and during antimicrobial therapy after rinsing the mouth with fresh tap water. The specimens were either processed immediately or refrigerated at 4 C for no longer than 1 hr. (iii) Unstimulated saliva specimens were obtained from subjects who had no clinical symptoms of lower respiratory disease.

2 9l-3 VOL. 18, 1969 QUANTITATION OF MICROORGANISMS IN SPUTUM 215 TABLE 1. Procedures for liquefaction, dilution, and were examined twice a week for 3 weeks. The bacteria culture ofsputuma and fungi which were recovered were identified by standard bacteriological and mycological methods (2). Dilution of liquefied sputum Inoculation of media A summary of the procedures for liquefaction, dilution, and culture of sputum is shown in Table 1. Specimen Dilu- Final Dilu- The number of viable microorganisms per milliliter Media D-ent dilution tion ofb sputum inoculated" of sputum was estimated by multiplying the number of colonies grown from a given tube in a dilution serief ml by the dilution factor of that tube and by the Liquefied (1:2) additional dilution resulting from the use of the.1- sputum (1. ml calibrated loop. Whenever possible, the calculations were performed on a dilution that yielded from ml) '- BA, CA, SDA, 3 to 1 colonies per plate. The total number of EMBA colonies of each organism was counted, and the number of viable organisms present per milliliter of spu- Dilution 1-1 (.1 ml) o-3 BA, CA tum was calculated for each different organism Dilution 1-3 isolated. (.1 ml) BA, CA Routine qualitative streak-plate cultures were Dilution1-X performed on some of the sputum specimens. A small (.1 ml) BA, CA area of a blood-agar plate was inoculated with a. sterile swab, first dipped into a purulent portion of the a Liquefaction procedure: using equal volumes specimen. This inoculated area was streaked over the of sputum and NAC solution, shake for 1 to 3 min remaining part of the plate with a flame-sterilized wire loop. The results were recorded qualitatively as the "predominant organism." Comparative studies. The following studies were performed by means of the materials and methods (1:2 dilution of sputum). 1 Inoculum:.1 ml. c Abbreviations: BA, blood-agar; CA, chocolate-agar; SDA, Sabouraud Dextrose Agar; EMBA, Eosin Methylene Blue Agar. Liquefaction and dilution of specimens. A 2% solution of N-acetyl-L-cysteine (NAC; Mead Johnson & Co., Evansville, Ind.) was prepared by dissolving 1 g of NAC in 43.5 ml of sterile, phosphate-buffered.85% NaCl and adding to this solution 6.5 ml of 1 N NaOH (final ph 7.2). The buffered NAC solution was stable for 3 to 4 days at 4 C. Contact with rubber or metal was avoided to minimize deterioration (14). Equal volumes of the 2% NAC solution and sputum were transferred to sterile screw-cap tubes containing four to six, 5-mm, washed glass beads and were agitated on a Vortex Jr. mixer for 1 to 3 min, depending on the viscosity of the specimen. Sterile, phosphate-buffered.85% NaCl was used as a diluent for preparing dilutions of 1-1, 1-s, 1-5, and 1-7 for the liquefied sputum. Cultivation techniques. The surface of the plates was inoculated by using a platinum and rhodium loop (1, 5) made of Brown and Sharpe no. 19 gauge wire, with an inside diameter of 4 i.3 mm, and calibrated to deliver.1 ml. Using such a flame-sterilized calibrated loop, one loopful of each well-mixed sputum dilution was inoculated onto appropriate media. To deliver the correct volume of the loop, it was held in a vertical position when being withdrawn from the specimen. The.85% NaCl dilutions were plated on the following media: Sabouraud Dextrose Agar (Difco), Eosin Methylene Blue Agar (EMB) (Difco) chocolate agar, and sheep blood-agar. The blood-agar plates and EMB plates were incubated aerobically at 37 C, and the chocolate-agar plates were incubated at 37 C under a carbon dioxide tension of approximately 2 to 3% in a candle jar. All plates were examined after incubation for 24 hr. The Sabouraud Dextrose Agar plates were incubated at 25 C and described above: (i) comparison between homogenized and nonhomogenized sputum in the recovery of different microbial species; (ii) comparison between homogenized specimens of aged sputum, fresh sputum, and saliva in the isolation rate of various microorganisms; (iii) quantitative estimation of populations of microorganisms present in homogenized specimens of aged sputum, fresh sputum, and saliva; (iv) quantitation of organisms in fresh sputum specimens collected before and after rinsing the mouth with fresh tap water in subjects who could produce a spontaneous sputum specimen on command; and (v) study of clinical pneumonia by using serial quantitative and qualitative sputum cultures before and after the institution of antimicrobial therapy. RESULTS Results of 54 sputum specimens cultured by both the routine streak-plate method and the quantitative method are shown in Table 2. Neisseria species, a streptococci, HaemopIilus, and Pseudomonas aeruginosa were isolated approximately the same number of times. Diplococcus pneumoniae and Klebsiella were isolated more often by the quantitative method (3% and 17%, respectively) than by the routine streakplate method (17% and 6%, respectively). Organisms isolated less frequently by the quantitative method than by the routine method were Staphylococcus epidermidis and Proteus. The number of times and the frequency an organism was recovered by the quantitative method from specimens of aged sputum, fresh sputum and saliva, as well as the number of specimens of each examined, are shown in Table

3 216 MONROE ET AL. APPL. MICROBIOL. TABLE 2. Comparison of results obtained by the liquefaction-dilution method and the routine streak-plate methodfor 54 sputum specimens Organism Routine streak- plate method Liquefactiondilution method No. of No. of times Per cent times Per cent isclated isolated Staphylococcus epidermidis Neisseria spp a Streptococci Diplococcus pneumoniae Haemophilus spp Klebsiella sp Proteus spp Pseudomonas aeruginosa Except for Neisseria species in the aged sputum specimens, the isolation rates among the organisms of the normal oral flora were very similar in three studies. D. pneumoniae was isolated 1% more often and Haemophilus was isolated 3% more often when the sputum was fresh than when it had been held at room temperature before culturing. Whether the sputum was fresh or aged appeared to make no difference in the recovery rates of Klebsiella and Candida. Certain organisms, namely D. pneumoniae, Haemophilus, and Escherichia coli, were not isolated from any of the saliva specimens. In general, there were fewer kinds of organisms isolated per specimen from aged sputum than from fresh sputum or saliva (Table 4). Except for Klebsiella and Haemophilus, larger populations of organisms were recovered from aged sputum than from fresh sputum and saliva. The mean numbers for those organisms which are considered to be members of the normal oral flora, i.e., S. epidermidis, Neisseria species, and et streptococci, were very similar for fresh sputum and saliva (Table 5). Comparative results of quantitative sputum cultures before and after the mouth was rinsed with fresh tap water are presented in Table 6. A total of six specimens were examined. No S. epidermidis, E. coli, or Candida were isolated TABLE 3. Comparision of the number of isolates from aged sputum specimens, fresh sputum specimens, and saliva specimens Aged sputum Fresh sputum Saliva (78 specimens)a (13 specimens) (2 specimens) Organism Times Per cent *Times Per cent Times Per cent isolated ssolated isolated Staphylococcus epidermidis Neisseria spp axstreptococci Diplococcus pneumoniae Haemophilus spp Klebsiella sp Enterobacter sp Escherichia coli Candida spp a Incubated at room temperature for approximately 4 hr or longer before being cultured. TABLE 4. Comparison of the number of species of microorganisms isolated per specimen from aged sputum, fresh sputum, and saliva No. of microbial species per specimen" Mean no. Median No. of ofseis no.of Type of specimen specimens specsmens -. ~~~~ per species per specimen Aged sputum (17%) 28 (36%) 25 (32%) 7 (9%) 5 (6%) Fresh sputum (11%) 27 (26%) 33 (32%) 2 (2%) 12 (11%) 3. 3 Saliva... 2 (%) 5 (25%) 7 (35%) 7 (35%) 1 (5%) a Number and frequency of specimens containing given number of microbial species. ~~~~~~~~~~~~~~~per

4 VOL. 18, 1969 QUANTITATION OF MICROORGANISMS IN SPUTUM 217 Organism TABLE 5. Quantitation of organisms in aged sputum, fresh sputum, and saliva (logio organisms/ml) Aged sputum Fresh sputum Saliva (78 specimens) (13 specimens) (2 specimens) Range Median Mean Range Median Mean Range Median Mean Staphylococcus epidermidis Neisseria spp a Streptococci Diplococcus pneumoniae Haemophilus spp Klebsiella sp Enterobacter sp Escherichia coli Candida spp TABLE 6. Quantitative results from sputum cultures before and after rinsing mouth with fresh tap water Before rinsing After rinsing Organism _ Per cent change Times Organisms/ml Times Organisms/ml isolated (mean) isolated (mean) Haemophilus spp X X 11-75% Diplococcus pneumoniae X X % a Streptococcus X X 15-93% Neisseria spp X X 17-78% Staphylococcus epidermidis X 17-1% Escherichia coli X 13-1% Candida spp X 13-1% TABLE 7. Comparison between quantitative and routine sputum cultures in pneumonia (before treatment) No. of times isolated Organism Quantitative Routine (17 or (predomgreater) inant organism)' Diplococcus pneumonia 7 4 Haemophilus spp Both of the above... 3 Staphlococcus aureus1... I Total isolated/total patients... 19/19 6/17 a Two patients had no routine sputum cultures on admission. after the mouth was rinsed, as compared with one isolate of each before rinsing. In three instances (Haemophilus, a streptococci, and Neisseria species), the number of times an organism was isolated remained the same before and after rinsing. However, D. pneumoniae was isolated three times before mouth rinsing but was found four times after rinsing, and was present in greater numbers. Even though some of the organisms were still present after rinsing the mouth, their numbers were significantly reduced. Populations of a streptococci and Neisseria species were decreased 93% and 78%, respectively, whereas the count of Haemophilus was decreased 75%. Quantitative cultures on admission sputum specimens from 19 patients with pneumonia revealed D. pneumoniae to be present in greatest numbers in seven patients, Haemophilus in eight, and both D. pneumoniae and Haemophilus present in largest numbers in three; in one patient, S. aureus was the most numerous (Table 7). These organisms in high numbers were recovered from all 19 patients by use of the quantitative method, whereas by the routine qualitative streak-plate method they were the predominating organisms in only 6 of 17, approximately 35 %. Routine streak-plate cultures on admission were not done on two patients. In all instances, the organisms noted above were present in the sputum specimens in counts of 17 organisms/ml

5 I. 156 ~~~~~~~~~~~~Li KEFLIN 4 GM/ D IV I MONROE ET AL. APPL. MICROBIOL. h. ~TETRACYCLINE GM D '... i _ 2 6 FIG. 1. Serial quantitative sputum cultures from a pneumonia patient prior to (day ) and during antimicrobial therapy. D. pneumoniae is the apparent pathogenic organism and is the only organism occurring in numbers greater than 17/ml. This level of apparent pathogenic significance is indicated by the horizontal line at that point. I Ln Io o 6 1i3 PENICILLIN 2.4 M.U./D IM 3 FIG. 3.IFSerial quantitative counts on sputum in pneumonia with two potential pathogens, H. parainfluenzae and D. pneumoniae. Day and line ofsignificance are as in Fig z v i I PENICILUN 2.MU. /D IV- i---ampicilun 6GM, cd IV --43C4 9-KAAKMYCIN GM/ D - 75 MGf #- D. PNEUMONIAE 19 ^_ A- A^H. INFLUENZAE P. AERUGINOSA I'1... KLEBSIELLA SP. 8 I.FCOUSTIN4 1 MG/ O71 1,6 FIG. 2. Serial quantitative sputum cultures from a patient with pneumonia apparently due to two organisms, D. pneumoniae and H. influenzae. Day and horizontal line ofsignificance are as in Fig. 1. or greater. None of the other organisms occurred in concentrations this high. Quantitative cultures were repeated at intervals during the patients' hospital treatment. These results on four patients are shown in Fig a.. a a a 9 1 II FIG. 4. Serial quantitative sputum cultures from a patient with pneumonia followed by superinfections with Klebsiella and P. aeruginosa. Day and line of significance are as in Fig. 1.

6 VOL. 18, 1969 QUANTITATION OF MICROORGANISMS IN SPUTUM 219 DISCUSSION Certain obvious differences were evident in the results of culturing the same sputum specimen by both the quantitative method and the routine streak-plate method. May (8) pointed out that to obtain accurate, viable culture results it was necessary to use techniques which took into account the irregular distribution of pathogens in sputum specimens. The quantitative method used in the present study produced a uniformly homogeneous specimen, and this homogeneity might have accounted for the higher isolation rates of D. pneumoniae and Klebsiella than were found by routine plating. It is possible that the capsule characteristic of these two organisms caused clumping and uneven distribution throughout the specimen. The investigations of Richardson and Jones (11) have shown that in saliva the quantitative range of S. epidermis is 1 to 14 organisms/ml. Since an.1-ml calibrated loop was used in the present study to streak plates from the serial dilution tubes, the number of organisms per milliliter in a sputum specimen would have to have been present in a minimal concentration of 13 organisms/ml to be observed (1-1 dilution tube X 1-2 calibrated loop). This fact would account for the smaller number of S. epidermidis isolates by the quantitative method than by the routine streak-plate method. There was no appreciable difference in the isolation rate of Neisseria species and a streptococci by the two methods. Their populations are reported to be greater than 14 organisms/ml (6, 11), and they would, therefore, be isolated as often by the quantitative method as by routine methods. The isolation rate of D. pneumoniae and species of Haemophilus from aged sputum was less than that of fresh sputum. May and Delves (9) compared the isolation rates of D. pneumoniae and H. influenzae from fresh specimens of sputum with those from specimens sent to the laboratory by mail. The isolation rates for both organisms from mailed specimens were approximately half those from fresh specimens. The results of the present study revealed that fewer kinds of organisms were isolated per specimen from aged sputum than from fresh sputum or saliva. The most plausible reason for this may be that certain fastidious organisms cannot remain viable for long periods of time outside their natural environment before being cultured on appropriate media. For example, certain organisms, such as the pneumococcus, often undergo autolysis. Also, sputum and saliva contain antimicrobial substances, such as lysozyme, which can destroy organisms, and some organisms produce substances which have antimicrobial properties toward other microbial species. These factors are possibly related to the decrease in the number of microbial species in aged sputum specimens. The fact that quantitative counts of various bacterial species in aged sputum were higher than those in fresh sputum or saliva indicates that the organisms continued to multiply after collection. Even though D. pneumoniae and H. influenzae had a tendency to be nonviable in the aged sputum specimens, when they were isolated their numbers were as high as those of some of the other organisms, such as S. epidermidis and a streptococci. This suggests that if the organisms are able to survive, they usually multiply. The results showed that rinsing the mouth with fresh tap water before obtaining a sputum specimen decreased the bacterial counts of the normal oral flora organisms. Organisms whose colony counts are reported to be low in saliva (11, 15), i.e., S. epidermidis, E. col, and Candida, were reduced below minimal numbers recognized by this method. As was noted previously, however, the number of D. pneumoniae isolates, as well as the quantitative count, was increased after mouth rinsing. Reasons for this increase are not clear. However, rinsing the mouth should reduce the concentration of any inhibiting substance in the saliva. Also, rinsing would reduce the populations of a streptococci, allowing D. pneumoniae to be recognized more easily, since their colonies are very similar in macroscopic appearance. Thus, if the mouth is rinsed before obtaining a sputum specimen to reduce the numbers of a streptococci, the possibility of confusing these colonies should be greatly diminished. The patients with pneumonia whose sputum yielded one organism in high numbers usually a 25 ui I- :52 ui 15 1= 1- b 1 z NON-PATHOGENS IPATHOGENS <2Li; mrl~ in 11 II IIM am.mm1 LOGIO ORGANISMS/ ML. FIG. 5. Quantitative distribution of pathogens and nonpathogens cultured from the sputum of 19 pneumonia patients before antimicrobial therapy.

7 22 MONROE ET AL. APPL. MICROBIOL. responded within a few days to appropriate antimicrobial therapy and had no problem with superinfection (Fig. 1). Pneumonia caused by two different organisms responded similarly when therapy was directed against susceptible organisms (Fig. 2). e. The results obtained from culturing serial sputum specimens from the 19 patients with pneumonia by using the quantitative method show that this method may provide better bacteriological data to the physician, thus leading to more effective patient care in lowerrespiratory-tract infections. The presence of potential pathogens in sputum may be ignored if they occur in low numbers, e. g., Candida in Fig. 1 and Enterobacter in Fig. 2. The administration of antimicrobial agents to eliminate these organisms from the sputum would have been useless and possibly detrimental to the patient. Quantitative counts appeared to establish which was the etiologic agent if two different potential pathogens were present in the sputum, e.g., Haemophilus and D. pneumoniae in Fig. 3. One patient died due to a superinfection first with Klebsiella followed by P. aeruginosa (Fig. 4). The clinical course of this patient might have been altered if the significance of the high colony counts of P. aeruginosa, indicating a true superinfection, had been realized sooner, and appropriate change of antibiotics had been made earlier. By this technique, the organism associated with a given lower-respiratory-tract infection, whether it be primary pneumonia or a superinfection, is present in concentrations of 17 or more organisms/ml of sputum. Normal mouth flora and opportunists are found in sputum before therapy in concentrations of 16 organisms/ml or less (Fig. 5). Quantitative cultures performed on serial specimens in identical fashion will reveal a decrease or disappearance of the pathogenic microorganisms in response to appropriate therapy and, moreover, may provide the earliest indication of the emergence of superinfection before there is any apparent worsening of the patient's clinical condition. Quantitative counts above 17 of normal flora organisms during antimicrobial therapy could not be related to superinfection in this study (Fig. 3). This study shows that quantitative microbial cultures of sputum provide the physician with useful information regarding infections of the lower respiratory tract and that they are a better guide than routine culture in the selection of the proper antibiotic to administer to the patient. ACKNOWLEDGMENTS This investigation was supported by Public Health Service research grant AI-7618 and training grant I-TI-AI-326 from the National Institute of Allergy and Infectious Diseases, by research grant CC-81 fiom the National Communicable Disease Center, and by research funds from the Veterans Administration Hospital, Oklahoma City, Okla. LITERATURE CITED 1. American Public Health Association Standard methods for the examination of dairy products, 11th ed. Amer. Publ. Health Ass., New York. 2. Bailey, W. R., and E. G. Scott Diagnostic microbiology, 2nd ed. C. V. Mosby Co., Saint Louis. 3. Brumfit, W., M. L. N. Willoughby, and L. L. Bromley An evaluation of sputum examination in chronic bronchitis. Lancet 2: Dineen, P The importance of the route of infection in experimental biliary tract obstruction. Surg. Gynecol. Obstet. Int. Abstr. Surg. 119: Hoeprick, P. D Culture of the urine. J. Lab. Clin. Med. 56: Kraus, F. W., and C. Gaston Individual constancy of numbers among the oral flora. J. Bacteriol. 71: Lapinski, E. M., E. D. Flakes, and B. C. Taylor An evaluation of some methods for culturing sputum from patients with bronchitis and emphysema. Amer. Rev. Resp. Dis. 89: May, J. R The bacteriology of chronic bronchitis. Lancet 2: May, J. R., and D. M. Delves The survival of Haemophilus influenzae and pneumococci in specimens of sputum sent to the laboratory by post. J. Clin. Pathol. 17: Okinaka, A. J., and P. Dineen Bacterial colony counts on bronchial washings. Ann. Surg. 167: Richardson, R. L., and M. Jones A bacteriologic census of human saliva. J. Dent. Res. 37: Sanford, J. P., C. B. Favour, F. H. Mao, and J. H. Harrison Evaluation of positive urine cultures. Amer. J. Med. 2: Shulman, J. A., L. A. Phillips, and R. G. Petersdorf Errors and hazards in the diagnosis and treatment of bacterial pneumonias. Ann. Int. Med. 62: Woodhams, A. W., and G. R. Mead A comparison between pancreatin and N-acetyl-L-cysteine as sputum liquefying agents for the culture of organisms. Tubercle 46: Young, G., H. G. Resca, and M. T. Sullivan The yeasts of the normal mouth and their relation to salivary acidity. J. Dent. Res. 3: