A Previously Undescribed Gram-negative Bacillus Causing Septicemia and Meningitis RAYMOND

A.

BOBO, P H . D . , AND ELEANOR

J.

NEWTON,

M.T. (ASCP)

Department of Clinical Pathology, University of Alabama in Birmingham, The Medical Center, Birmingham, Alabama

ABSTRACT

T h e unidentified Gram-negative bacillus showed susceptibility to all antimicrobials tested except gentamicin. (Key words: Sepsis and meningitis caused by unidentified bacillus; Unidentified bacillus causing septicemia and meningitis.) BACTERIUM most frequently associated with dog and cat bites is Pasteurella multocida.1'3 This paper describes a case of septicemia and meningitis associated with dog bites and produced by an organism that cannot be classified using current taxonomic schemes. T h e etiologic agent is similar to 15 other isolates reported to the Communicable Disease Center in Atlanta, Georgia, over the past several years. Dog bites or association with dogs was a common

THE

occurrence in patients from which the organisms were isolated (personal communication, R. E. Weaver, Center for Disease Control, Atlanta, Georgia). A complete description of the agent, which may represent a new disease entity, is presented.

Received April 15, 1975; received revised manuscript J u n e 11, 1975; accepted for publication J u n e 11, 1975. 564

Report of a Case T h e patient, a 42-year-old Negro man, initially came to the emergency room because of seizures. He had been in good health until the evening before admission, when he had had a severe frontal headache. On the morning of admission, while

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Bobo, Raymond A., and Newton, Eleanor J.: A previously undescribed Gram-negative bacillus causing septicemia and meningitis. Am J Clin Pathol 65: 564-569, 1976. This report describes a case of septicemia and meningitis secondary to dog bites by two different dogs on two consecutive days. The case is noteworthy because of the unusual characteristics of the etiologic agent and the inability to place the etiologic agent into any currently defined genus or to identify it by the existing systems of classification.4 T h e organism is a small, thin, Gram-negative bacillus after 24 hours of incubation on blood agar; after prolonged incubation, it becomes filamentous. The organism is catalase- and oxidase-positive, hydrolyzes esculin, and forms acid in glucose, xylose, and maltose after 21 days' incubation. T h e organism does not manifest lysis on sheep blood agar, and does not grow on MacConkey, Salmonella-Shigella, Centrimide, nutrient, or Kligler iron agars. T h e tests for urea, nitrate reduction, and indol are negative.

April 1976

SEPSIS CAUSED BY UNIDENTIFIED BACTERIUM CULTURES CSFt-t* NEG BLOOD + * + + + + ANTIMICROBIAL AGENTS

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at home, he had suffered a grand mal seizure, with fecal and urinary incontinence. After 15 minutes he had become alert, but he had had a similar seizure half an hour later. He was brought to the hospital, where he had his third grand mal seizure. A veterinarian employed the patient, and it was learned that seven days prior to admission, while he was on the job, a dog had bitten him, and the following day he had been bitten by a second dog. Although both bites, located on the hands, had broken the skin, the wounds had healed well, with no sign of infection. A review of systems and family history were noncontributory. As a child the patient had had seizures which were probably related to febrile episodes; however, none had occurred since 4 years of age. Seven years before admission the diagnosis of pulmonary tuberculosis had been made, and he had subsequently spent 3 years in sanitariums. All cultures for Mycobacterium tuberculosis had been negative for the past 5 years, and he had taken no antituberculosis therapy for 4 months prior to ad-

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mission. He was known to be a heavy consumer of alcohol and had been drinking quite heavily until two days before he was admitted to the hospital. No episode of delirium tremens had occurred according to both the patient and his mother. On physical examination, the patient was acutely ill and semicomatose, thrashing around and moving all four extremities. T h e temperature was 104.4 F., rectally, pulse 130 per min., respiratory rate 18 per min., and blood pressure 132/70 mm. Hg. T h e r e was no evidence of jaundice, petechiae or lymphadenopathy. T h e neck, though supple to rotation, did offer resistance to anteroposterior flexion, it was thought that the patient had positive Brudzinski's and Kernig's signs. A few scattered rhonchi were present throughout the lung fields, and slightly decreased breath sound could be heard over the right lobe posteriorly. Examination of the abdomen disclosed no abnormality. Over the metacarpophalangeal joints of both the left and right hands were several healed abrasions, which, according to the patient's

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Fie. 3 (lower). Gram stain of bacilli taken from sheep blood agar plate after incubation for 6 days in 10% C 0 2 atmosphere, x 1,000.

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mother, were the locations of the dog bites. Except for the patient's sensorium, the neurologic examination was interpreted as normal.

Laboratory studies disclosed an initial leukocyte count of 8,900 with 49% neutrophils, 32% band forms, 17% lymphocytes, 2% monocytes; hematocrit 36%;

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FIG. 2 (upper). Gram stain of bacilli taken from sheep blood agar plate after 72 hours of incubation of 10% CO z atmosphere, x 1,000.

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Table 1. Cellular and Colonial Morphology of Bacterium I. Cellular morphology Shape Axis Size Sides End Pleomorphic forms Staining

Edge Internal Colony surface Optical characteristics Consistency

Punctate 0.5 mm. on blood agar at 48 hr. Circular Convex Entire Granular Smooth and glistening Translucent Butyrous

platelet count 272,000. The BUN on admission was 9 mg. per 100 ml. and creatinine 1 mg. per 100 ml. Urinalysis disclosed no abnormality except 1 + protein and 5 erythrocytes per high-power field. A lumbar puncture showed 19 leukocytes per cu. mm., with a differential count of 30% neutrophils and 70% lymphocytes, protein 103 mg. per 100 ml. and glucose 85 mg. per 100 ml. (serum glucose 145 mg. per 100 ml.). Gram stain, acid-fast stain, and India ink preparation of the cerebrospinal fluid obtained on admission were negative. Nine hours later a repeat lumbar puncture yielded a grossly clouded fluid, with an opening pressure of 300 mm. H 2 0 , closing pressure 150 mm. H 2 0 , and 2,300 leukocytes per cu. mm., with a differential count of 96% neutrophils and 4% lymphocytes, protein 722 mg., and glucose less than 25 mg. per 100 ml. (serum glucose 147 mg. per 100 ml.). At this time Gram stain of the fluid revealed small Gram-negative bacilli and numerous leukocytes. All of the initial spinal fluid specimens grew out a small pleomorphic Gram-negative bacillus. Six blood cultures drawn on the day of admission yielded identical organisms. Acid-fast stains and acid-fast cultures of the urine and sputum were negative. Chest

x-ray was essentially within normal limits except for a right upper lobe infiltrate which was compatible with inactive tuberculosis. On admission the initial impression was Hemophilus influenzae meningitis. T h e patient was treated with ampicillin, gentamicin, chloramphenicol, and carbenicillin. T o cover the possibility of tuberculous meningitis, isoniazid, ethambutal, and steroids were given. By the second hospital day the patient appeared alert and was afebrile; he felt much better except for a frontal headache. T h e organism showed susceptibility to all antimicrobials tested except gentamicin. All antibiotics were discontinued except ampicillin, given intravenously, with which the patient was treated for 14 days. Lumbar puncture on the fourteenth hospital day revealed complete clearing except for a protein concentration of 112 mg. per 100 ml. The patient was afebrile and asymptomatic when discharged (Fig. 1). Discussion T h e organism was initially isolated from both spinal fluid and blood on a 5% human blood agar plate cross streaked with

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II. Colonial morphology Size Shape Surface elevation

Predominantly bacillary after 48 hours on blood agar-pleomorphic forms increased after 72 hours at 37 C. Straight with some curved forms Small, short to medium length ( 1 - 3 /am.), thin rods Parallel, no spores formed Pointed, tending to round with age After 72 hours at 37 C. most were fairly uniform in shape and size, some coccobacillary forms. With age bacteria tended to become filamentous, with some branching and more pronounced curving in individual cells Evenly Gram-negative

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Table 2. Biochemical Characteristics of the Organism No lysis

?

-t

+t

-

+ + -

No growth No growth

No growth No growth

+ + + —

Trace

+ /+ —

* Cystine trypticase agar base (BBL). t After 7 days of incubation at 37 C , tubes covered witb sterile mineral oil. t After 21 days of incubation at 37 C, tubes covered with sterile mineral oil.

Staphylococcus aureus and incubated at 37 C. in an atmosphere of 10% C 0 2 . On primary isolation the organism produced mucoid growth on blood agar, but it lost this characteristic after serial passage. A tentative identification of the organism was that of an encapsulated Hemophilus species. This identification was changed after subsequent examination showed the organism did not require either X or V factor. Gram stains of the organism from both the blood culture and spinal fluid revealed the appearance shown in Figures 2 and 3. Although the organism did not re-

quire blood for growth, it did require an enriched medium such as brain-heart infusion broth or trypticase soy broth. Nutrient agar alone was not sufficient for growth. T h e organism grows at both 37 and 25 C. on an enriched medium, but would not grow at 45 C. Cellular and colonial morphology and biochemical characteristics are presented in Tables 1 and 2. The utilization of all carbohydrates was negative after the first seven days of incubation; however, glucose, xylose and maltose degradation with the production of acid could be demonstrated at the end of 21 days' incubation. After 72 hours at 37 C. on a sheep blood agar plate most of the organisms were fairly uniform in size and shape, with some coccobacillary forms. T h e organisms stained evenly Gram-negative; however, with continued incubation the bacillus tended to become quite filamentous, manifesting both branching and curving in individual cells (Fig. 3). T h e biochemical characteristics of catalase and oxidase positivity, negative growth on MacConkey and Kligler iron agars, negative tests for nitrate reduction, indol and urea, together with the production of acid in glucose, xylose, and maltose, separates this bacterium from presently described genera. Thus, this isolate could not be placed in any of the existing characterized groups of bacteria, nor could it be identified by any of the currently used classification schemes. 1,4 Bacilli possessing the characteristics described in this report have not been previously described as pathogenic. Cultures of material from the oral cavities of the dogs that had bitten the patient were negative for the bacterium described in this report. Thus, the reservoir of the agent remains unknown. Lee and Buhr cultured Pasteurella multocida from 17% of lesions following dog bites; however, no other Gram-negative bacilli were identified.

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Action on blood Gas from glucose Oxidative or fermentative CTA base* With 1% glucose With 1% xylose With 1% mannitol With 1% sucrose With 1% maltose With 1% lactose 10% glucose 10% lactose Catalase Oxidase MacConkey SS Centrimide Trypticase soy agar Nutrient agar Simmons citrate Christensen urea Nitrate Indol Kligler iron agar slant 1 butt H 2 S-Paper MR/VP Gelatin Litmus milk Motility Esculin hydrolysis Lysine decarboxylase Arginine dihydrolase Ornithine decarboxylase

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SEPSIS CAUSED BY UNIDENTIFIED BACTERIUM

Although dog bites were associated with this case, this may be coincidence. It is possible that this organism may be part of the normal flora of the human oral cavity and act as an opportunistic pathogen in individuals who have predisposing illnesses or defective defense mechanisms.

also examined this isolate, but were unable to classify the organism.

References 1. Blair JE, Lennette EH, Truant JP: Manual of Clinical Microbiology. Second edition. Washington, D.C., American Society for Microbiology, 1974, pp 246-249 2. Holloway VVJ, Scott EG, Adams YB: Pasteurella multocida infection in man. Report of 21 cases. Am J Clin Pathol 51:705-708, 1969 3. Lee J L H , Buhr AJ: Dog bites and local infection with Pasteurella septica. Br Med J 1:169171, 1960 4. Weaver RE, Tatum HW, Hollis DC: T h e Identification of Unusual Pathogenic Gram Negative Bacteria. U.S. Department of Health, Education and Welfare, Communicable Disease Center, Atlanta, Georgia, 1972

Erratum T h e r e was an e r r o r in T a b l e 7 of t h e article, " S t a g i n g in t h e T h e r a p y of C a n c e r o f t h e Breast," which a p p e a r e d o n p a g e 7 6 2 of t h e D e c e m b e r 1975 issue of t h e American Journal of Clinical Pathology. At t h e b o t t o m of t h e table, u n d e r t h e h e a d i n g "Sinus histiocytosis in axillary l y m p h n o d e s , " t h e scores s h o u l d be listed as o n e for " p r e s e n t " a n d two for "absent," r a t h e r t h a n t h e reverse.

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Acknowledgments. T h e following individuals contributed to this report: Donald M. Taft, M.D., Department of Clinical Pathology, Marguerite Burgin and Eleanor J. Newton, Bacteriologists, Department of Clinical Pathology, University of Alabama in Birmingham. The Center for Disease Control, Atlanta, Georgia, and the bacteriology laboratories of the Alabama Department of Public Health

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