Vol. 59, No. 4INFECTION AND IMMUNITY, Apr. 1991, p. 1352-13580019-9567/91/041352-07$02.00/0Copyright C 1991, American Society for Microbiology

Pathophysiology of Citrobacter diversus Neonatal Meningitis:Comparative Studies in an Infant Mouse Model

ALEJANDRO L. SORIANO, 2t ROBERT G. RUSSELL,2'3 DAVID JOHNSON,4 ROSANNA LAGOS,2tIANCU SECHTER,5 AND J. GLENN MORRIS, JR.2*

Division of Neonatology, Department of Pediatrics,' Division of Geographic Medicine, Department of Medicine, andCenter for Vaccine Development,2 Department ofPathology and Program of Comparative Medicine,3 and Division of

Infectious Diseases, Department of Medicine,4 University of Maryland School of Medicine, Baltimore, Maryland 21201,and Government Central Laboratories, Ministry of Health, Jerlusalem, Israel5

Received 7 November 1990/Accepted 21 January 1991

Citrobacter diversus is a cause of devastating neonatal meningitis, with illness characterized by formation ofmultiple brain abscesses. We developed an infant mouse intracranial inoculation model to evaluate thepathophysiology of C. diversus neonatal infections. Eighteen of 26 strains inoculated intracranially at a dose ofca. 3.3 x 103 CFU caused >50% mortality in 2-day-old mice. No correlation was seen between theepidemiologic characteristics of a strain and its rate of mortality. When seven C. diversus isolates (four isolatesfrom patients with meningitis, three from non-central nervous system [CNS] sites) were further evaluated,mortality was significantly correlated with bacteremia. The initial lesion in the CNS was a suppurativeventriculitis beginning 1 to 2 days postinoculation. Subsequent ventriculomegaly was associated withventriculitis and periventricular abscessation. Brain lesions were seen with all strains, although strains of lowvirulence (as measured by having no bacteremia and low mortality) caused less-severe damage. An age-relatedsusceptibility to C. diversus brain lesions was demonstrated, with 5-day-old mice showing a significantreduction in, and 8-day-old mice being apparently resistant to, infection and CNS damage. Our data indicatethat C. diversus has a propensity to cause abscess formation in the neonatal mouse brain, with characteristicpathologic findings; however, the factors that determine whether a strain will cause meningitis in a humaninfant remain to be identified.

Citrobacter diversus is a gram-negative bacillus that is arare but devastating cause of neonatal meningitis, occurringsporadically or in epidemics (5, 10, 12, 19, 22, 25, 31, 32).The fatality rate of patients with meningitis is reported to be34%, with 90% of survivors having various degrees of mentalretardation (11, 13). In contrast to patients with otherbacterial causes of neonatal meningitis such as Escherichiacoli K-1 strains and group B Streptococcus strains, 77% ofC. diversus meningitis patients develop brain abscesses (11).Although the clinical and epidemiologic features of C. diver-sus infections in neonates have been well described, we lacka good understanding of the associated virulence mecha-nisms. The importance of putative virulence factors (16) indevelopment of disease remains to be determined, and therole of host factors, such as age, is unclear. The pathogenicmechanisms involved in cerebral abscess formation havealso been controversial (7, 15, 17).To assess the pathophysiology of C. diversus neonatal

meningitis, we and others have investigated a number ofpossible animal models (17, 23a); we report here resultsobtained after intracranial inoculation of the bacterium intoinfant mice. With our infant mouse model we sought todetermine whether there were differences among strains inpathogenicity and whether these differences correlated withepidemiologic background or with standard markers such asserotype, biotype, plasmid profile, or outer membrane pro-tein (OMP) profile. We were also interested in evaluatingprogression of the central nervous system (CNS) lesions and

* Corresponding author.t Present address: Trover Clinic, Madison, KY 42431.t Present address: Hospital Roberto del Rio, Santiago, Chile.

determining whether there was an age-related susceptibilityto infection in infant mice, as has been described in humans(11, 22).

MATERIALS AND METHODS

Bacterial strains. Twenty-six isolates of C. diversus wereexamined (Table 1). This includes 11 isolates from cases ofneonatal meningitis, 3 isolates associated with bacteremia,and 12 isolates cultured from other sites. The organismswere obtained from different geographic locations within theUnited States and Israel. E. coli K-12 strain HB101 was usedas a negative control. All isolates were stored at -70°C in Lbroth with 15% glycerol.

C. diversus isolates were serotyped for both 0 and Hantigens at the Government Central Laboratories, Jerusa-lem, Israel, by previously described methods (1). Biotypingwas based on the fermentation of dulcitol, rhamnose, su-crose, and sorbose (26). Plasmid profiles were determined byusing an alkaline plasmid extraction technique (2). Forselected strains, OMPs were extracted and prepared byusing a modification of the method described by Sears andRichardson (21, 28). Strains for OMP extraction were grownin L broth for 16 h at 37°C; proteins were visualized bysodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) by using discontinuous gels (30) with stackingand resolving gels of 4% (pH 6.9) and 11% (pH 8.7),respectively.

Intracranial challenge of neonatal mice. The study wasconducted in four parts. A preliminary experiment wasconducted to establish a dose-response curve and to deter-mine an appropriate inoculum size for subsequent studies.Two-day-old mice were challenged with C. diversus 52

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TABLE 1. Characteristics of 26 C. diversus strains studied and their respective mortality outcome

Strain Source Biotype Plasmid profilea Serotype Mortalityb

Meningitis-associated strains52 Meningitis/Maryland E I 3:a:2 16/21 (76)4511 Meningitis/Kentucky D XX 3:a:- 9/9 (100)836 Meningitis/Maryland E I 3:a:2 11/11 (100)4408 Meningitis/Louisiana A XXI 2:a:2 12/12 (100)4406 Meningitis/Delaware E Il 3:a:2 12/12 (100)666-546 Meningitis/Israel D XXII 3:a:2 15/15 (100)19266 Meningitis/Maryland E II 3:a:2 11/11 (100)4409 Meningitis/Texas E XXIII 2:a:2 5/40 (13)6947 Meningitis/Maryland A II 5:i:- 6/9 (67)3078 Meningitis/Texas A XXIV 2:a:2 3/12 (25)871-3851 Brain abscess/Israel E XXV 3:a:2 4/10 (40)

Septicemia-associated strains22525 Blood/Maryland E 1I 3:a:- 8/10 (80)53114 Blood/Maryland C II 15:a:2 8/8 (100)4963 Blood/Oklahoma E II 1:a:- 6/18 (33)

Strains isolated from other sites20871 Cervix/Maryland C II 15' 13/13 (100)896-109 Vagina/Israel C II 15:a:2 11/11 (100)26357 Rectum/Marylandd C XV 15C 10/10 (100)30488 Wound/Maryland E II 3:a:2 11/11 (100)14545 Breast/Maryland E II 4/10 (40)75 Rectum/Marylandd C XXVI 11:a:2 9/9 (100)745-72 Eye/Israel A XXVII 1:c:- 5/13 (38)27447 Rectum/Marylandd C XIII 15:a:2 11/11 (100)26359 Umbilical cord/Marylandd C XVIII 15' 10/10 (100)663-972 Eye/Israel D XXVIII 12:a:2 4/11 (40)896-2744 Urine/Israel A IV 2:a:2 3/11 (27)2742 Hands/Marylandd.e E I 3:a:2 11/11 (100)a Designation of plasmid profile follows that used previously by Morris et al. (22); strains with profiles that had not been previously identified were numbered

consecutively, beginning with XX.b Number of pups that died/number tested. Numbers in parentheses represent percent mortality.' Data on flagellar antigen type not available.d Asymptomatic patients.e Strain associated with meningitis outbreak.

(isolated from an infant with meningitis during a majornursery outbreak in Baltimore [19]) in doses of 101 to 107CFU in 10-fold serial dilutions; each dilution was adminis-tered to 10 mice from a single litter.Experiment 1 was subsequently undertaken with all 26 C.

diversus isolates to determine the mortality rate with eachstrain in 2-day-old infant mice. In experiment 2, we selectedseven isolates from experiment 1 to evaluate bacteremia andbrain pathology in 2-day-old mice. Experiment 3 evaluatedthe effects of age on mortality and the development of CNSlesions in 5- and 8-day-old pups by using C. diversus 52.

Challenge organisms were prepared by inoculating singlecolonies from an overnight Luria agar (L agar) plate into Lbroth and incubating the cultures for 18 h at 37°C. Bacterialconcentrations of the inocula were determined by relatingoptical density at 600 nm to CFU per milliliter; all determi-nations were confirmed by plate count assays done induplicate.

Pathogen-free pregnant (12 to 14 days gestation) femaleCD-1 mice were obtained from Charles River BreedingLaboratories. The dams were examined twice daily to detectbirths. The time of day (morning or late afternoon) thatlitters were subsequently challenged with the inoculum at 2,5, or 8 days old corresponded to whether pups were deliv-ered during the day or overnight so that the actual age oflitters differed by no more than 12 h at the time of challenge.All mice in a litter were inoculated with the same strain (or,

in dose-response studies, with the same dilution of the samestrain) and were then returned to their natural mothers. Oneto three litters were inoculated with each strain or dilution ineach of the experiments. Bacteria in a volume of 100 p.1 wereinoculated intracranially into the right parietal area by pen-etrating the skull with a 27-gauge needle attached to a 1-mltuberculin syringe. The site of inoculation was 3 mm behindthe right eye and 2 mm from the sagittal suture. In allexperiments, inoculations were performed by the same

investigator (A.L.S.). For studies of mortality, animals wereobserved for a maximum of 7 days.

Blood cultures and histopathology. Blood cultures wereobtained at the time of sacrifice by cardiac puncture after theskin was disinfected with povidone and then with 70%isopropyl alcohol. Samples (100 ,ul) of blood were inoculatedonto L agar plates. Identity of isolates from positive bloodcultures was confirmed by use of the API system (AnalytabProducts, Plainview, N.Y.).For pathologic examination, mice were decapitated and

the brains were collected and fixed in 10% neutral bufferedFormalin. Coronal sections of the brain were processed forparaffin embedding. The 5-p.m sections were stained withhematoxylin and eosin. Histologic interpretation was under-taken by one investigator (R.G.R.) who was blinded to theidentification of the isolate and the age of the mouse at thetime of inoculation and sacrifice. To histologically evaluatethe extent of the inflammation and damage in the brain

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1 10 100 1000 10000 100000 1000000 10000000inoculum size (CFU)

FIG. 1. Results of initial dose-response studies with C. diversus52, showing percent mortality as a function of inoculum size. Tenmice from a single litter were inoculated with each dilution tested.

lesions and to assess sampling variability, serial sectionswere prepared from the brains of selected mice with repre-sentative lesions.

RESULTS

Preliminary dose-response studies with C. diversus 52. Theresults of the preliminary experiment using intracranialchallenge of strain 52 in 2-day-old mice are shown in Fig. 1.The dose of 3.3 x io3 used in all subsequent experimentswas the calculated 85% lethal dose.Experiment 1: mortality in 2-day-old mice (26 isolates).

After intracranial inoculation in 2-day-old mice, 15 of the 26C. diversus isolates caused 100% mortality, 3 isolates causedmortality of 67 to 80%, and the remaining 8 isolates hadmortalities of 6 to 40% (Table 1). There was no correlationbetween mortality and the site from which the isolate wasrecovered (CNS versus blood versus other sites), the geo-graphic location from which the isolate was obtained, or theplasmid profile of the isolate (Tables 1 and 2). Strains of

TABLE 2. Mortality outcome of 26 C. diversus isolates byepidemiologic characteristics and source

Epidemiologic characteristic Mortality in:or source <50%5o >50o

BiotypeA 3 2C 0 7D 1 2E 4 7

Serogroup01 2 002 3 103 1 905 0 1011 0 1012 1 0015 0 6Unknown 1 0

SourceCNS 3 8Other sites 5 10

TABLE 3. Rates of mortality and bacteremia for sevenC. diversus isolates

Strain Source Bac- Mortalityb

Meningitis-associatedstrains

52 Meningitis/Maryland 6/6 21/21 (100)19266 Meningitis/Maryland 3/5 12/12 (100)4409 Meningitis/Texas 0/6 2/14 (14)6947 Meningitis/Maryland 4/5 7/8 (88)

Strains isolated fromother sites

896-109 Vagina/Israel 3/4 12/12 (100)75 Rectum/Maryland 3/5 15/15 (100)663-972 Eye/Israel 0/6 0/8 (0)a Number of pups with bacteremia/number tested. Results are from animals

sampled on days 2, 4, and 6.b Number of pups that died/number tested. Numbers in parentheses repre-

sent percent mortality. As these experiments were conducted separately fromthe initial studies of mortality, there are differences in the reported mortalityrates, compared with those shown in Table 1.

biotype C, serotype 15, were significantly more likely tocause 100% mortality (P = 0.01; Fischer's exact test).However, none of the biotype C, serogroup 15 strains werefrom patients with meningitis.

Experiment 2: mortality, bacteremia, and brain histopa-thology in 2-day-old mice (seven isolates). (i) Bacteremia andmortality. Seven C. diversus isolates were selected forfurther study (Table 3). Mortality studies were repeated forthese isolates. Results were comparable to those obtained inexperiment 1: strains 52, 19266, 6947, 896-109, and 75 causedhigh mortality, while minimal mortality was seen with strains4409 and 663-972 (Fig. 2). When analyzed statistically,significant heterogeneity in mortality rates was observedamong litters inoculated with the same strain for strains 52and 663-972. For strain 52, one litter (inoculated as part ofexperiment 1) had 6 deaths of 11 infants, while the remaininglitters tested had 10 deaths of 10 infants (experiment 1), 13deaths of 13 infants (experiment 2), and 8 deaths of 8 infants(experiment 2) (P < 0.001, G test for heterogeneity). Forstrain 663-972, there were 4 deaths of 11 infants (experiment1) and 0 deaths of 8 infants (experiment 2) (P = 0.025, G testfor heterogeneity). However, even with this heterogeneity,mortality rates for litters inoculated with the high mortality

52 19266 4409 6947 896-109 75 663-972strain number

FIG. 2. Cumulative mortality (%) in 2-day-old mice, by day afterinoculation, for seven selected C. diversus strains. Symbols: _,days 1 to 2; 1 , days 3 to 4; , days 5 to 6.

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FIG. 3. Response of 2-day-old mouse 2 days postinoculationwith C. diversus 896-109. Moderate numbers of neutrophils arepresent in the lateral ventricles. The ependyma is intact. Hematox-ylin and eosin stain. Magnification, x950.

strain 52 were significantly higher than rates for littersinoculated with low mortality strain 4409 (P = 0.03) or663-972 (P = 0.04, t test using Arcsin transformed data).

Additional pups were sacrificed on days 2, 4, and 6postinoculation, and blood samples were obtained for cul-ture. Bacteremia, if present, was always detected on day 2postinoculation; only animals inoculated with strain 52 werestill bacteremic by day 6. The two strains that did not causebacteremia (4409 and 663-972) were the two strains thatcaused minimal mortality; a significant correlation was seenbetween overall rates of bacteremia and mortality (r = 0.8, P= 0.01; Spearman correlation coefficient).

(ii) Brain histopathology. On days 1 to 2, mice inoculatedwith 4409 and 663-972 (the two low mortality strains) hadlocalized acute necrosis of the cortex at the site of inocula-tion without infiltration of neutrophils or abscessation. Miceinoculated with strains 52, 19266, 6947, 896-109, and 75 (thehigh mortality strains) had acute ventriculitis with numerousneutrophils in the lateral ventricles (Fig. 3) and suppurativemeningitis. In addition, mice inoculated with strains 19266and 6947 developed severe purulent ventriculitis with ab-scessation, destruction of the ependyma, ventriculomegaly,and infiltration of neutrophils into the adjacent parenchymawith formation of bilateral periventricular abscesses (Fig. 4).Bacteria were located intracellularly and extracellularly.On days 3 to 4 postinoculation, there were no lesions in

the brains of mice inoculated with strain 663-972. Miceinoculated with strain 4409 showed unilateral abscessationinvolving the lateral ventricle and expanding locally into theadjacent parenchyma. This was associated with destructionof the ependyma and ventriculomegaly. In contrast, miceinoculated with the other five strains (52, 896-109, 19266,6947, and 75) showed severe bilateral abscessation involvingthe ventricles and expanding into the adjacent parenchymaaccompanied by ventriculomegaly. In addition to abscessa-tion, strains 52 and 6947 also caused extensive adjacentcortical damage with edema, malacia, and cavitation (Fig. 5).On days 5 to 6 postinoculation, mice inoculated with strain

663-972 had localized abscessation involving the cortex and

.,.FIG. 4. Response of 2-day-old mouse 2 days postinoculation

with C. diversus 6947. Marked ventriculomegaly is seen, withdestruction of the ependyma and extension of the severe purulentinflammation into the periventricular brain parenchyma. There iswell-demarcated brain abscessation (arrows). Hematoxylin andeosin stain. Magnification, x300.

ventricles (no abscessation was found in mice challengedwith this strain and examined at earlier time intervals). Nobrain lesions were found in surviving mice inoculated withstrain 4409. There were no survivors from the group of miceinoculated with strain 896-109. The mice inoculated withthree (19266, 6947, and 75) of the remaining four strainsshowed well-demarcated periventricular abscesses withearly glial scarring at the perimeter (Fig. 6). The brains ofmice inoculated with strain 52 showed severe purulentexudate in the ventricles, extensive cortical damage andabscessation accompanied by destruction of the ependyma

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FIG. 5. Response of 2-day-old mouse 3 days postinoculationwith C. diversus 52. Malacia and neutrophil infiltration in thecerebral cortex are present adjacent to abscessation. Hematoxylinand eosin stain. Magnification, x950.

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FIG. 6. Response of 2-day-old mouse 6 days postinoculationwith C. diversus 6947. Glial scar formation (arrows) is present at theperimeter of a well-demarcated brain abscess in the ventricles andadjacent parenchyma. Hematoxylin and eosin stain. Magnification,x950.

and necrosis, and large numbers of intracellular and extra-cellular bacteria (Fig. 7). Adjacent to the abscess there wasmalacia and gliosis without evidence of early glial scarring.The severity of meningitis was variable in the animals

examined. In some mice, no meningitis was observed. Inothers, mild to moderate suppurative meningitis involved thearea immediately overlying the abscess and was both dorsaland dependent in the brain stem and cerebellar areas.Except for focal malacia at the site of inoculation, no brain

lesions were noted in the control mice inoculated with sterilemedia or E. coli HB1O1 in doses as high as i07 CFU. Theability of the different strains to cause bacteremia, mortality,and CNS lesions did not correlate with the presence of any

A

FIG. 7. (A) Large numbers of intracellular bacteria (arrowheads)and (B) extracellular bacteria (arrows) in the brain of a 2-day-oldmouse 6 days postinoculation with C. diversus 52. The mouse had a

severe purulent ventriculitis with abscessation and malacia in theadjacent parenchyma. Hematoxylin and eosin stain. Magnification,x 5,000.

7:VE~~

FIG. 8. Response of an 8-day old mouse 4 days postinoculationwith C. diversuis 52 showing a localized meningeal abscess. Hema-toxylin and eosin stain. Magnification, x300.

specific OMP band on SDS-PAGE or with any one plasmidor plasmid profile (data not shown).Experiment 3: mortality and brain histopathology in 5- and

8-day-old mice inoculated with C. diversus 52. (i) Mortalityand bacteremia. Six (26%) of 23 mice (composing two litters)inoculated with strain 52 at the age of 5 days died. Themortality rates among these litters were significantly lowerthan the rates among litters inoculated with this strain at theage of 2 days (P = 0.012; t test using Arcsin transformeddata). Bacteremia was evaluated in six mice inoculated at theage of 5 days; only one was bacteremic.There was no mortality (14 mice) or bacteremia (3 mice)

among mice inoculated with strain 52 at the age of 8 days.Mortality rates among litters inoculated at the age of 8 dayswere significantly lower than those among litters inoculatedat the age of 2 days (P = 0.008; t test using Arcsintransformed data).

(ii) Brain histopathology. At 2 days postinoculation of5-day-old mice with strain 52, there was marked bilateralsuppurative ventriculitis and ventriculomegaly with largenumbers of bacteria. In contrast to the extensive abscessa-tion and malacia observed in 2-day-old mice on days 3 to 6postinoculation, the mice inoculated at the age of 5 daysshowed localized necrosis in the superficial cortex at the siteof inoculation accompanied by diffuse moderate meningitisat days 3 to 4 postchallenge. The focal lesion was healingwith scarring on days 5 to 6 postinoculation, and there wasmeningitis with infiltration of low to moderate numbers ofneutrophils and mononuclear cells. As in 2-day-old mice, nobrain lesions other than focal malacia at the site of inocula-tion were seen in 5-day-old mice inoculated with E. ccliHB101 or sterile culture media.Mice inoculated at the age of 8 days examined on day 2

postinfection had localized cortical necrosis at the site ofinoculation. On day 4 after inoculation there was a unilaterallocalized abscess in the meninges which did not extend intothe brain parenchyma (Fig. 8). At 6 days postinfection, the

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PATHOPHYSIOLOGY OF C. DIVERSUS NEONATAL MENINGITIS 1357

local lesion at the site of inoculation was healing with earlyscar tissue.

DISCUSSION

In this study we describe a mouse model in which absces-sation was produced in the brain after intracranial inocula-tion of low doses of C. diversus. No CNS lesions were seenin animals inoculated with sterile media or with E. coliHB101, emphasizing the etiologic role of C. diversus.

In prior work in our laboratory we were unsuccessful indeveloping models for C. diversus by using intraperitonealinoculation of adult mice (with and without iron loading),subcutaneous inoculation of infant rats, and oral inoculationof infant mice (23a). Kline et al. (17) reported an infant ratmodel for C. diversus meningitis by using simultaneousintraperitoneal and intranasal inoculation. We found this tobe cumbersome, requiring large numbers of animals (ca. 90infants per strain to show a statistically significant differencein mortality between the two strains in Kline's study) and anexcessively large inoculum size to produce infection. Thehistopathologic changes of purulent ventriculitis and adja-cent parenchymal abscessation documented in this rat modelwere similar to the brain lesions that we observed in ourinfant mouse model. Our model also appears to mimic thehuman infection in several important respects: abscessationoccurred only in the periventricular areas, there was a lackof involvement of the brain stem and cerebellum, andage-related susceptibility was demonstrated.

In investigations of outbreaks of C. diversus neonatalmeningitis there has been a suggestion that certain strains (asidentified by biotype, serotype, plasmid profile, and/or chro-mosomal restriction endonuclease digest) possess increasedvirulence; this suggestion was made on the basis of theobservation that only one strain of several present in aneonatal nursery was associated with occurrence of disease(10, 11, 22). Our data indicate that there are clear differencesin virulence among C. diversus strains after CNS inocula-tion. Despite some litter-to-litter variability, it was possibleto demonstrate statistically significant differences in mortal-ity rates between high and low mortality strains. In oursecond experiment (seven strains) we found that the twostrains with low mortality rates produced no bacteremia andhad only localized abscessation, in contrast to the severeabscessation and malacia observed with the more virulentstrains. This is in keeping with the work by Kline et al. (17),in which differences in mortality, bacteremia, and occur-rence of meningitis were noted when two strains (one from achild with meningitis and the other from an apparent trachealcolonization) were compared.While we found that strains differed in virulence in our

model, we were unable to correlate virulence with the sourceof the isolate (i.e., CNS versus blood versus other sites).This may be a function of the relatively small number ofstrains included in the study or may reflect difficulties inusing data on source of isolation to characterize clinicalvirulence of strains (i.e., all strains isolated from an asymp-tomatic person may not be avirulent). Alternatively andperhaps more likely, there may be factors other than viru-lence after CNS inoculation that determine whether a C.diversus strain will cause disease in a human infant.

It has not been possible to consistently associate menin-gitis cases with any particular C. diversus serogroup orbiotype (12). While strains of biotype C, serotype 15, weremore likely to kill mice in our model, none of these isolateswere from human patients with meningitis. Previous inves-

tigators have suggested that the occurrence of disease isassociated with the presence of a 32- or 69-kDa OMP (16,34); this suggestion was made on the basis of differencesnoted between strains from persons with and without men-ingitis. In our model (in contrast to the work by Kline et al.[16, 17]) we saw no association between the presence of anyOMP and patterns of mortality or CNS lesions.

In our histopathologic studies, the development of lesionsin 2-day-old mice was initiated by purulent ventriculitiswhich caused destruction of the ependyma, exposing theperiventricular parenchyma to bacterial invasion and subse-quent abscessation. Depending on the virulence of thestrain, the abscesses became delineated by early glial scar-ring or progressed into extensive cortical damage. Thedescriptions of brain pathology in human neonates infectedwith C. diversus include brain abscessation and necrosiswith hemorrhage in the white matter of the cerebral hemi-spheres (7, 8, 18). One author suggested that the hemorrhageand malacia in the white matter were due to septic vasculitis(7). Our observation of the absence of vasculitis and throm-bosis suggests that infarction does not play a role in thepathogenesis of the abscess in the infant mouse model.Although hemorrhage was not seen in the brains of the infantmice, the mice inoculated with some strains had severemalacia indicative of the propensity for destructive necrosisby C. diversus. Cerebral hemorrhage in human neonatesmay reflect species differences in the response of the neona-tal brain to injury or may be a consequence of the route ofinoculation. The lack of involvement of other areas of thebrain may indicate that C. diversus has a tropism for theperiventricular area or the ependymal layer, as has beendescribed for E. coli (24), and one could speculate that C.diversus recognizes specific binding sites in the neonatalmouse brain. Further studies with this model may help todefine the pathogenic mechanism(s) involved.

In this study we looked at the chronological developmentof brain lesions beginning 2 days after inoculation in 2-, 5-,and 8-day-old mice. The severity of disease was age related,with absence of bacteremia and with lower mortality andlower numbers of mice with brain abscessation in 5- and8-day-old mice than with 2-day-old mice. The brain histologyin the older mice showed localization of the ventriculitis andmeningitis without marked destruction of the periventriculararea by abscessation. The reason for this age-related suscep-tibility to C. diversus infection is not known. Possibleexplanations could include a change in the biochemicalcharacteristics, postnatal neuroanatomical development, orhost defense capabilities of the infant mouse brain between 2and 5 days of age (6, 9, 14, 24). Neonatal animals andhumans have been shown to have impaired host defensemechanisms, such as diminished levels of complement,profound abnormalities in phagocytic function, and defi-ciency in immunoglobulin M and immunoglobulin A andsome classes of immunoglobulin G (14, 20). Experiments tomanipulate and enhance host defenses have been done withgroup B Streptococcus strains and Listeria monocytogenesby using gamma globulin, neutrophil transfusions, andgamma interferon (3, 4, 27). Further studies are needed toassess the relevance of such interventions in C. diversusinfections.

In summary, our intracranial inoculation model provides ameans for evaluating progression of intracranial lesionsassociated with C. diversus infections. Because of the directroute of inoculation, this model cannot be used to addressquestions relating to the initial route of infection (althoughuse of scalp electrodes, which has been noted in some

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outbreak reports [10, 19], might create a situation analogousto direct inoculation in some human infants). Results in themodel do not correlate with the clinical source of the strain(i.e., whether a strain was isolated from a patient withmeningitis), further suggesting that occurrence of illness inhuman infants is dependent on factors other than virulenceof the organism after CNS inoculation. However, the modeldoes appear to be of value in assessing pathophysiologicmechanisms involved in C. diversus CNS infections and may

help to provide some understanding of why this and othergram-negative organisms (29, 33) cause such devastatingbrain lesions in human neonates.

ACKNOWLEDGMENTS

We thank Steve Wasserman for statistical assistance.This work was supported in part by a grant from the University of

Maryland Medical Biotechnology Center.

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2. Birnboim, H. C., and J. Doly. 1979. A rapid alkaline extractionprocedure for screening recombinant plasmid DNA. NucleicAcids Res. 7:1513-1523.

3. Chen, Y., A. Nakane, and T. Minagawa. 1989. Recombinantmurine gamma interferon induces enhanced resistance to Liste-ria monocytogenes infection in neonatal mice. Infect. Immun.57:2345-2349.

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