Vol. 33, No. 3INFECTION AND IMMUNITY, Sept. 1981, p. 939-9430019-9567/81/090939-05$02.00/0

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Growth of the Pittsburgh Pneumonia Agent in Animal CellCultures

CHARLES R. RINALDO, JR.,'2* A. WILLIAM PASCULLE,12 RICHARD L. MYEROWITZ,1,2FRANCIS M. GRESS,2 AND JOHN N. DOWLING'3

Department ofMicrobiology,l* University of Pittsburgh Graduate School of Public Health, andDepartments ofPathology and Medicine,3 Presbyterian- University Hospital and University of Pittsburgh

School ofMedicine, Pittsburgh, Pennsylvania 15261

Received 20 February 1981/Accepted 11 June 1981

Pittsburgh pneumonia agent (Legionella micdadei) grew in monkey, chicken,and human cell cultures. Pittsburgh pneumonia agent grew predominantly in thecytoplasm, resulting in a nonfocal, mild cytopathic effect.

Pittsburgh pneumonia agent (PPA) is a re-cently identified, fastidious, gram-negative bac-terium that has been associated with severepneumonitis in immunosuppressed patients (8,10). PPA resembles Legionella pneumophilaand other Legionella-like bacteria in its pheno-typic characteristics (5, 9). The names Legion-ella pittsburgensis (9) and Legionella micdadei(4) have consequently been proposed for thisorganism.The intimate interaction of PPA with host

cells appears to be of significance in the patho-genesis of and host response to PPA infection.The bacilli are frequently seen within cytoplas-mic vacuoles of polymorphonuclear leukocytesand macrophages of lung tissue (3). In this in-vestigation, an in vitro model was developed forthe study of the interrelationships of PPA withhost cells.PPA (strain EK) was isolated in 6-day-old

embryonated hen's eggs from the lung tissue ofa renal transplant recipient with acute purulentpneumonia (9), and the titers were determinedas the number of colony-forming units (CFU) onbuffered charcoal-yeast extract agar (10). Vero(African green monkey kidney) and HeLa (hu-man epitheloid cervical carcinoma) cells wereobtained from the American Type Culture Col-lection, Rockville, Md. Human foreskin cellsoriginated from primary tissue obtained fromMagee Women's Hospital, Pittsburgh, Pa., andwere used during passages 9 to 25. The cellswere maintained at 370C in closed bottles withEagle miznimum essential medium (GIBCO Lab-oratories, Grand Island, N.Y.) supplementedwith 10% heat-inactivated fetal calf serum, twicethe standard concentration of vitamins and es-sential amino acids, 2 mM glutamine, the stand-

ard concentration of nonessential amino acids,24 ,ug of nystatin per ml, and 0.8 mg of sodiumbicarbonate per ml. Chicken embryo (CE) cellswere prepared by treatment of 11-day-old CEswith a 0.6% trypsin solution in ethylenediamine-tetraacetic acid. The CE cells were maintainedwith Eagle miniimum essential medium supple-mented with 6% heat-inactivated newborn calfserum, twice the standard concentration of vi-tamnins, 25 ,ug of nystatin per ml, and 0.8 mg ofsodium bicarbonate per ml.

Subcultures of Vero, HeLa, human foreskin,and CE cells were grown to confluency in 35-mm plastic dishes with and without 22-mm2glass cover slips for 48 h (Vero, HeLa, and hu-man foreskin cells) or for 72 h (CE cells) at 37°Cin 5% CO2 and humidity. Cell monolayers wereinfected with various concentrations of PPA (0.1ml per dish) diluted in Hanks balanced saltsolution (GIBCO) containing 2 mg of sodiumbicarbonate per ml. After a 1-h adsorption pe-riod at 37°C in 5% C02, 2 ml of fresh mediumwas added to each plate; uninfected animal cellswere included as controls in each experiment.All cell cultures were examined on each samplingday for contamination by subculture to sheepblood agar and thioglycolate and brain heartinfusion broths.At various time intervals, the cytopathic effect

of PPA infection was monitored by direct lightmicroscopic examination of the unstained cellcultures and by modified (1% H2SO4 in alcohol)Ziehl-Neelsen acid-fast staining of cover slipsfrom the cell culture plates. Replicate, infectedcell cultures were scraped into the medium witha rubber policeman and frozen at -700C. Forassay of PPA, the samples were thawed, soni-cated (2 min at 140 W; Raytheon Sonic Oscilla-

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tor; Raytheon, Waltham, Mass.), and inoculatedonto buffered charcoal-yeast extract agar (0.1 mlof 10-fold dilutions in Hanks balanced salt solu-tion per 100-mm plate). Bacterial colonies werecounted after 4 days of incubation at 37°C. Incertain experiments, PPA-infected Vero cellswere fixed for electron microscopic studies witha 3% glutaraldehyde solution while still attachedto the culture dishes as previously described (3).The cells were scraped from the dishes, postfixedwith osmium tetroxide, embedded in epoxyresin, sectioned, and stained before electron mi-croscopic examination.

Initial studies showed that confluent mono-layers of Vero cells (approximately 106 cells per35-mm plate) supported the growth ofPPA afterinfection at various input multiplicities (CFU/Vero cell ratios, 0.01, 0.001, and 0.0001). Similarpeak titers of PPA (4.2 to 4.8 log1o CFU/ml)were detected by 7 days post-inoculation at allinput multiplicities. In nine subsequent experi-ments, peak titers of PPA have ranged from 5.2to 7.2 log1o CFU/ml by 7 to 9 days in Vero cellsinfected at a low input multiplicity (0.0001; rep-resentative experiment, Fig. 1). A similar patternof growth was noted in PPA-infected humanforeskin cells (data not shown). PPA did notgrow in fresh culture medium alone (Fig. 1) orin conditioned medium with or without lysedVero cells. Infected Vero cells did not display avisible cytopathic effect by light microscopy dur-ing the first 5 days of infection. By 7 days, therewere small separations in the monolayer andincreased amounts of floating debris. At 14 days,approximately 95% of the Vero cell monolayers

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infected at high multiplicities (0.01 and 0.001)were destroyed; monolayers infected at the low-est multiplicity (0.0001) exhibited increased sep-arations between cells and floating debris, butwere still intact (Fig. 2).PPA also grew in CE cells to peak titers of

approximately 4 to 5 logio CFU/ml by 7 days ofinfection (input multiplicity, 0.0001; representa-tive experiment, Fig. 1). In contrast to Vero,

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FIG. 1. Growth of PPA in Vero (A), CE (O), andHeLa (0) cell cultures at an input multiplicity of0.0001 and absence ofgrowth in cell culture mediumalone (A).

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FIG. 2. Cytopathic effect ofPPA in Vero cell cultures at 14 days of infection. (A) Control, uninfected Verocell monolayer; (B) 14 days after infection with PPA (input multiplicity, 0.0001). Bar = 25 mn.

INFECT. IMMUN.

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FIG. 3. Thin sections of Vero cells infected with PPA (input multiplicity, 0.0001) for 4 (A) and 14 (B) days.(A) Vero cells infected for 4 days containing organisms with an electron-lucent (L) and electron-dense (D)periplasmic space; (B) Vero cells infected for 14 days containing only organisms with an electron-denseperiplasmic space. Bar = 0.5 ,um.

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942 NOTES

human foreskin, and CE cells, levels of PPA inHeLa cells only increased from 1.2 to 1.7 log10CFU/ml on day 0 to peak titers of 2.2 to 2.5 logioCFU/ml by 5 days of infection (representativeexperiment, Fig. 1). Infected CE and HeLa cellsdisplayed a -75% cytopathic effect by 7 daysand a complete cytopathic effect by 14 days.However, uninfected, control cell monolayershad a comparable cytopathic effect at thesetimes due to aging in the culture (data notshown).The interaction of PPA with Vero cells was

examined further by light microscopy and elec-tron microscopy. At 2 days postinfection (inputmultiplicity, 0.0001), Vero cells did not containdetectable intracellular bacteria. Only non-acid-fast, extracellular bacteria were observed bylight microscopy; by electron microscopy, PPAbacilli, with an electron-dense layer in the peri-plasmic space, were also seen extracellularly.These cells were presumably part of the inocu-lum. By 4 to 5 days of infection, a small numberof Vero cells contained clumps of non-acid-fastbacteria. Numerous non-acid-fast organisms anda rare acid-fast bacterium were observed in thecell culture medium. Approximately 5% of theVero cells contained intracellular bacteria byelectron microscopic analysis. Infected cells gen-erally displayed a few large or many small cy-toplasmic vacuoles enclosing from 1 to morethan 20 bacterial cells (Fig. 3). Approximately80% of the organisms had an electron-lucent

periplasmic space, although certain cells con-tained nearly equal numbers of bacteria withand without the electron-dense periplasmicspace. Only cells without this layer were ob-served in forms suggestive of division.On 7 and 9 days postinfection, the Vero cell

monolayers contained large numbers of predom-inantly non-acid-fast bacteria in the cell cyto-plasms (Fig. 4) and in the culture medium. Acid-fast organisms were also occasionally seen withinthe cytoplasms and culture fluids. Approxi-mately 50 to 70% of the Vero cells containedPPA with and without the electron-dense peri-plasmic layer, including dividing forms, at 9days. Cellular remnants were observed enclosingnumerous bacteria. At 14 days, some of the Verocells were lysed, but the remaining intact cellscontained predominantly non-acid-fast and afew acid-fast organisms. By electron microscopy,the bacteria appeared to be mostly bacilli withthe electron-dense periplasmic layer (Fig. 3).Organisms lacking this layer were relativelyscarce and were detected in only 20% of infectedcells.The growth characteristics of PPA in vitro

resemble those recently reported for L. pneu-mophila (1, 12). Both organisms appear to growintracellularly and cannot be propagated in cellculture medium alone. They grow in cells de-rived from several animal species, although cer-tain cell lines (HeLa) do not support the growthof PPA as well as others (Vero). PPA and L.

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FIG. 4. Vero cells infected for 7 days with PPA and stained by the modified Ziehl-Neelsen technique.Organisms are seen intracytoplasmically as a single bacillus (arrowheads) and large masses of bacilli(arrow). Bar = 2.5 ,um.

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VOL. 33, 1981

pneumophila also do not grow on most types ofartificial medium, but can be cultivated onbuffered charcoal-yeast extract and charcoal-yeast extract agar (5, 9). Thus, the ability ofthese bacteria to grow intracellularly is similarto that of facultative intracellular bacteria suchas brucellae (6), mycobacteria (11), and salmo-nellae (7).PPA is an unusual gram-negative organism in

that it is weakly acid-fast by the modified Ziehl-Neelson stain (10). We have recently describedtwo ultrastructural variants of PPA, includingcells with and without an electron-dense layer inthe periplasmic space (3). We have attempted todelineate whether the acid-fast property of PPAcould be related to either type of morphologicalvariant. No correlation was apparent in our stud-ies of the kinetics of growth of PPA in Vero cellcultures. In most experiments, few acid-fast or-ganisms were noted throughout the course ofinfection (14 days), whereas the relative numberof cells with the electron-dense layer varied from20 to 80%. However, the acid-fast property doesappear to correlate with its growth in cells andanimals, since PPA grown on artificial mediumis not acid-fast (9). It is possible that the acid-fast characteristic of PPA is related to its viru-lence in the living host.

Infection of animal cell cultures with PPAmay serve as a model for further studies of theinteraction of PPA with host cells. PPA mayproduce toxins, similar to L. pneumophila (1, 2),that are demonstrable in cell cultures. PPA-in-fected cell cultures may also provide a sensitivesystem for examination of the effects of variousantibiotics on the intracellular growth of PPA.In this regard, preliminary investigations in ourlaboratories have indicated that PPA is suscep-tible to certain antibiotics (penicillin and genta-micin) in infected Vero cell cultures.

We thank Annie McCoy and Mary White for technicalassistance. This work was supported in part by a grant from

NOTES 943

the Samuel and Emma Winters Foundation, Pittsburgh, Pa.,and Public Health Service grant AI 17047 from the NationalInstitute of Allergy and Infectious Diseases.

LITERATURE CITED

1. Daisey, J., H. Friedman, C. Benson, M. Chesler, andJ. McKitrick. 1980. Intracellular growth of, and toxinproduction by, Legionella pneumophila in tissue cul-ture, p. 998-1000. In J. D. Nelson and C. Grassi (ed.),Current chemotherapy and infectious disease. Ameri-can Society for Microbiology, Washington, D.C.

2. Friedman, R. L., B. H. Iglewski, and R. D. Miller.1980. Identification of a cytotoxin produced by Legion-ella pneumophila. Infect. Immun. 29:271-274.

3. Gress, F. M., R. L. Myerowitz, A. W. Pasculle, C. R.Rinaldo, Jr., and J. N. Dowling. 1980. The ultrastruc-tural morphology of "Pittsburgh pneumonia agent."Am. J. Pathol. 101:63-77.

4. Hebert, G. A., A. G. Steigerwalt, and D. J. Brenner.1980. Legionella miedadei species nova; classificationof a third species of Legionella associated with humanpneumonia. Curr. Microbiol. 3:255-258.

5. Hebert, G. A., B. M. Thomason, P. P. Harris, M. D.Hicklin, and R. M. McKinney. 1980. "Pittsburghpneumonia agent": a bacterium phenotypically similarto Legionellapneumophila and identical to the Tatlockbacterium. Ann. Intern. Med. 92:53-54.

6. Holland, J. J., and M. J. Pickett. 1956. Intracellularbehavior of Brucella variants in chick embryo cells intissue culture. Proc. Soc. Exp. Biol. Med. 93:476-479.

7. Kihlstrom, E. 1977. Infection of HeLa cells with Salmo-nella typhimurium 395 MS and MR10 bacteria. Infect.Immun. 17:290-295.

8. Myerowitz, R. L., A. W. Pasculle, J. N. Dowling, G.J. Pazin, M. Puerzer, R. B. Yee, C. R. Rinaldo, Jr.,and T. R. Hakala. 1979. Opportunistic lung infectiondue to "Pittsburgh pneumonia agent." N. Engl. J. Med.301:953-958.

9. Pasculle, A. W., J. C. Feeley, R. J. Gibson, L. G.Cordes, R. L. Myerowitz, C. M. Patton, G. W. Gor-man, C. L. Carmack, J. W. Ezzell, and J. N. Dowl-ing. 1980. Pittsburgh pneumonia agent: direct isolationfrom human lung tissue. J. Infect. Dis. 141:727-732.

10. Pasculle, A. W., R. L. Myerowitz, and C. R. Rinaldo,Jr. 1979. New bacterial agent of pneumonia isolatedfrom renal-transplant recipients. Lancet ii:58-61.

11. Shepard, C. C. 1955. Phagocytosis by HeLa cells andtheir susceptibility to infection by human tubercle ba-cili. Proc. Soc. Exp. Biol. Med. 90:392-396.

12. Wong, M. C., E. P. Ewing, Jr., C. S. Callaway, andW. L. Peacock, Jr. 1980. Intracellular multiplicationof Legionella pneumophila in cultured human embry-onic lung fibroblasts. Infect. Immun. 28:1014-1018.

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