APPLIED MICROBIOLOGY, Feb. 1968, p. 315-319Copyright © 1968 American Society for Microbiology

Vol. 16, No. 2Prinited int U.S.A.

Association of Mucoid Encapsulated Moraxella duplexvar. nonliquefaciens with Chronic Bronchitis

EDWARD BOTI7ONE AND JONA ALLERHAND

Department of Pathology, Mount Sinai Hospital Services, City Hospital Center at Elmhurst,Elmhurst, New York 11373

Received for publication 23 October 1967

The recovery of two strains of highly mucoid encapsulated Moraxella duplex var.

nonliquefaciens from the sputum of two patients suffering from chronic bronchitisis described. The biochemical, morphological, and pathogenic characteristics ofthis microorganism are discussed.

Members of the genus Moraxella, notably M.lacunata and M. liquefaciens, have long beenrecognized primarily for their eye pathogenicity(7). M. duplex var. nonliquefaciens may also pro-duce a mild conjunctivitis. In diseases other thanthose affecting the eye, M. duplex var. nonlique-faciens has been isolated particularly when itoccurs in the mucoid form.

Henriksen (1) described a mucoid strain of M.duplex var. nonliquefaciens that he recovered fromthe sputum of a 55-year-old laborer sufferingfrom bronchitis and bronchopneumonia. Murrayand Truant (5) reported the isolation of a similarmucoid strain from a throat culture that theyidentified according to Henriksen's criteria.Kaffka (4) also reported the isolation of twomucoid strains from healthy carriers and onenonmucoid strain from a 73-year-old patientsuffering from bronchopneumonia. Peel (6) iso-lated a mucoid strain from the sputum of a 76-year-old male patient suffering from chronicbronchitis.

This report concerns the predominant isolationof two highly mucoid encapsulated strains of M.duplex var. nonliquefaciens from sputum and theevaluation of these mucoid variants as potentialpathogens.

CLINICAL STUDIES

Case no. 1. An 84-year-old white male was admittedto the City Hospital Center at Elmhurst on 23 Feb-ruary 1967 because of severe dyspnea and cyanosis.The patient presented with a 50-year history of chronicbronchitis and a 10-year history of emphysema. Hehad a chronic cough with copious, purulent sputumaccompanied by frequent attacks of dyspnea. Hisadmission diagnosis was chronic bronchitis withrecent superimposed acute infection. Temperature atadmission was 99 F (37.22 C).

Laboratory tests showed that hemoglobin levelwas 11.7 g/100 ml and leukocyte count was 14,800,

with a shift to the left. The purified protein derivative(PPD) skin test for tuberculosis was negative, aswere the smear and culture of the sputum for tuber-cle bacilli. X-ray examination showed moderateemphysema and no evidence of a pulmonary neo-plasm. Sputum cultures revealed the predominantgrowth of large mucoid colonies (Fig. 1), which weresubsequently identified as M. duplex var. nonlique-faciens, mucoid form. A few colonies of Aerobacteraerogenes, Escherichia coli, and a-hemolytic strepto-cocci were also present. The organism proved sus-ceptible in vitro, by the disc agar-diffusion method,to many antibiotics (Table 1).The patient was placed on tetracycline twice a day.

After 24 hr, sputum cultures still showed the persist-ence of the same microorganism, but cultures becamenegative at 48 and 72 hr after admission. Sputum pro-duction diminished, probably as a result of the anti-microbial therapy, and the patient was discharged on6 March 1967, 11 days after admission. Tetracyclinetreatment was continued, and the patient was referredto the pulmonary clinic for followup.

Case no. 2. A 61-year-old white male was admittedto City Hospital Center at Elmhurst on 5 July 1967with a chief complaint of anorexia, chronic cough,and weight loss. The admission findings were chronicbronchitis and emphysema, cirrhosis of the liver dueto chronic alcoholism, abdominal distress, andanemia.

Laboratory tests showed that hemoglobin level was11.1 g/100 ml and leukocyte count was 6,900. X-rayexamination of the lungs showed old fibrotic calcifi-cations from tuberculosis treated 10 years previously.Bacteriological examination of the sputum failed toreveal the presence of tubercle bacilli. However,routine cultures showed the predominant growth ofmucoid M. duplex var. nonliquefaciens, accompaniedby a few colonies of Staphylococcus aureus, pneumo-cocci, Haemophilus influenzae, nonhemolytic strepto-cocci, and Escherichia coli. The mucoid organismproved susceptible in vitro, by the disc-agar diffusionmethod, to a variety of antimicrobial agents (Table1).The patient was placed on ampicillin, 250 mg four

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BOTIONE AND ALLERHAND

Injection of 1 ml of the sputum from case 1into the peritoneal cavity of two white miceproved nonlethal after 48 hr. However, when I mlof pure culture isolates from the above cases con-

s.

FiG. 1. Blood-agar plate showing growth of mucoidcolonies of Moraxella duplex var. nonliquefaciens.

TABLE 1. Antimicrobial susceptibility of the isolatedmucoid Moraxella duplex var. nonliquefaciens

c^9a= t g e e = ~~~~~~~~~~'4 a0Case. -

0 0

pg Ag pg pg units Ag pg

1 5a 5 2 2 2 5 2 Rb R2 5 5 2 2 2 5 2 R R

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FIG. 2. Direct smear of sputum. Many encapsulateddiplobacilli can be seen.

antibiotic giving

times a day, which resulted in a marked decrease inthe number of Moraxella colonies that developedfrom a 24-hr post-therapy sputum culture. At 48 hrafter the initiation of antimicrobial therapy, thesputum culture became negative for this microor-ganism.

The patient improved, gained weight, and was sub-sequently discharged with referral to the gastroin-testinal clinic for the followup of a bilateral diver-ticulosis discovered by X-ray examination.

REsULTSIn the two cases studied, the direct Gram

smears of the sputum revealed the presence ofnumerous gram-negative, markedly encapsulatedcoccobacilli with squared ends (Fig. 2 and 3). Asthe microscopic morphology resembled that ofKlebsiella, direct quellung reactions were per-formed with specific antisera, with negativeresults.

FIG. 3. India ink preparation from 24-hr blood-agarculture, demonstrating large capsules.

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FIG. 4. Smear of 24-hr blood-agar culture showing the marked pleomorphism. Irregular stained, vacuolated,andfilament forms are seen.

taining approximately 600 million organisms(MacFarland no. 2) were injected into the peri-toneal cavity of white mice, lethal results wereobtained in two out of four mice in case 1, andin all three mice in case 2. The organisms wererecovered from the heart blood of all mice thatexpired.The organisms grew well on blood- and choco-

late-agar, particularly under 10% CO2 tension,but, as contrasted to Klebsiella, they failed togrow on MacConkey or Endo agar. They werenitrate and oxidase positive, penicillin sensitive,and lacked either oxidative or fermentative powerswhen grown in Cystine Trypticase Agar (BBL) orphenol red agar base containing 1% dextrose,sucrose, lactose, maltose, mannitol, and xylose.After 24 hr, Gram-stained smears of culturesgrown on blood-agar plates showed markedpleomorphism ranging from typical gram-nega-tive coccobacilli with square ends to large irregu-lar stained filaments. Vacuolation and involutionforms were common (Fig. 4). According toHenriksen (2), these morphological and bio-chemical characteristics are typical of this micro-organism. Table 2 shows the biochemical charac-teristics observed by various investigators with

mucoid M. duplex var. nonliquefaciens. Thedifferences between this microorganism and Kleb-siella are obvious.

DIscUSSIONThe mucoid strains isolated from the sputum

were pathogenic for white mice when massivedoses of the organism were injected into theperitoneal cavity. The observation of Henriksenand Kaffka (1, 4), that only the mucoid strainsare pathogenic for mice, has been confirmed byour studies. However, in our cases, as in the casesof Henriksen and Peel, the mucoid strains re-covered were from symptomatic patients, whereasKaffka recovered his strains from healthy carriers.

Henriksen (3) recovered 99 isolates of M.duplex var. nonliquefaciens from 875 nose cultures,all nonmucoid and nonpathogenic for white mice.In our laboratory, 50 isolates of the same non-mucoid organism were recovered from a variety ofclinical sources (eye, nose, throat, sputum, blood,etc.); none of these strains proved pathogenic towhite mice, even in massive doses. It seems thatthe presence of increased amounts of capsularmaterial is paramount for the elicitation of mousepathogenicity.

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MORAXELLA FROM CHRONIC BRONCHITIS

As pointed out by Henriksen, rare recovery inthe laboratory of this microorganism may beattributed to (i) its CO2 and moisture require-ments for growth, and (ii) its morphologicalsimilarity to Klebsiella and occasionally to Neis-seria, which may confuse identification. However,pleomorphism, when observed, is a particularcharacteristic of this microorganism, which mayaid in identification.The possible etiological role of this organism

in chronic bronchitis is problematical. Its pre-dominance in the sputum culture and the con-comitant therapeutic response upon eliminationof these organisms by appropriate antibiotictherapy does suggest a possible etiological signifi-cance.

ACKNOWLEDGMENTSThe encouragement and support of W. Mautner

and S. S. Schneierson are greatly appreciated. Mrs. M.Dominguez provided invaluable technical assistance.

LITERATURE CITED1. HENRIKSEN, S. V. 1951. Moraxella duplex var.

nonliquefaciens as a cause of bronchial infection.Acta Pathol. Microbiol. Scand. 29:258-262.

2. HENRIKSEN, S. V. 1952. Moraxella: classificationand taxonomy. J. Gen. Microbiol. 6:318-328.

3. HENRIKSEN, S. V. 1958. Moraxella duplex var.nonliquefaciens, habitat and antibiotic sensi-tivity. Acta Pathol. Microbiol. Scand. 43:157-161.

4. KAFKA, A. 1955. Zum Vorkommen und zurPathogenitat von Moraxella duplex var. non-liquefaciens. Zentr. Bakteriol. Parasitenk. 164:451-455.

5. MuRRAY, R. G. E., AND J. P. TRUANT. 1954. Themorphology, cell structure and taxonomic af-finities of the Moraxella. J. Bacteriol. 67:13-22.

6. PEEL, J. A. 1966. Moraxella duplex var. nonlique-faciens in sputum. J. Med. Lab. Technol. 23:101-102.

7. TOPLEY, W. W. C., AND G. S. WILSON. 1964.Principles of bacteriology and immunity, p.1093-1103. The Williams and Wilkins Co.,Baltimore.

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