Nosocomial Pneumonia Caused by a Glucose-Metabolizing Strain of

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JOHN M. BOYCE,'* MAX R. TAYLOR,' E. BRUCE MITCHELL, JR.,2 AND JOAN S. KNAPP3. Departments ofMedicinel and Clinical Laboratory Sciences,2 ...
JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 1985, p. 1-3

Vol. 21, No. 1

0095-1137/85/010001-03$02.00/0 Copyright © 1985, American Society for Microbiology

Nosocomial Pneumonia Caused by a Glucose-Metabolizing Strain of Neisseria cinerea JOHN M. BOYCE,'* MAX R. TAYLOR,' E. BRUCE MITCHELL, JR.,2 AND JOAN S. KNAPP3 Departments of Medicinel and Clinical Laboratory Sciences,2 University of Mississippi Medical Center, Jackson, Mississippi 39216,1 and Neisseria Reference Laboratory, Department of Medicine, University of Washington, Seattle, Washington 981953 Received 29 May 1984/Accepted 15 September 1984

We describe what appears to be the first reported case of nosocomial pneumonia caused by Neisseria cinerea. The isolate metabolized glucose when tested in BACTEC Neisseria Differentiation Kits (Johnston Laboratories), but did not produce detectable acid in cystine-Trypticase (BBL Microbiology Systems) agar medium or in modified oxidation-fermentation medium. Clinical laboratories that rely on the BACTEC method for differentiation of pathogenic neisseriae should be aware of the fact that N. cinerea may mimic N. gonorrhoeae when tested in BACTEC Neisseria Differentiation kits. The ability of N. cinerea to grow well on tryptic soy and Mueller-Hinton agars and its inability to grow on modified Thayer-Martin medium are characteristics which help to distinguish N. cinerea from N. gonorrhoeae. In 1906, von Lingelsheim (11) described Micrococcus cinereus. The organism was described as being morphologically similar to Micrococcus (Neisseria) catarrhalis. It was renamed Neisseria cinerea by Murray in 1939 (9). Subsequent studies have confirmed that N. cinerea may colonize the oropharynx and, less commonly, the genital tract (4, 12; J. S. Knapp, P. A. Totten, B. H. Totten, and E. W. Hook III, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, C26, p. 316). A few infections which were probably caused by N. cinerea have been reported, but in each case the organism was misidentified as N. flavescens, N. gonorrhoeae, or Branhamella catarrhalis (1, 4, 10). In this report, we describe what we believe to be the first reported case of nosocomial pneumonia caused by a confirmed N. cinerea isolate. Although N. cinerea is considered to be asaccharolytic, the strain recovered in this case was glucose positive when tested in the BACTEC Neisseria Differentiation Kit (Johnston Laboratories, Cockeysville, Md.) and might have been misidentified as N. gonorrhoeae on the basis of the BACTEC results. Clinical microbiologists who use the BACTEC system for presumptive identification of pathogenic neisseriae should be aware of the fact that N. cinerea may give positive glucose readings in this system.

chial breath sounds over the left lower lobe. A Gram stain of endotracheal secretions showed many granulocytes, gramnegative diplococci, and a few gram-positive cocci. A culture of the endotracheal secretions grew predominantly a Neisseria species, which was interpreted as normal flora. Two blood cultures done with the BACTEC system were sterile. Erythromycin, 500 mg every 6 h, and gentamicin, 70 mg every 8 h, were begun. There was no improvement after 48 h, and his temperature rose to 104°F (40°C). Repeat examination of endotracheal secretions revealed many granulocytes and many gram-negative diplococci. Repeat culture of the secretions yielded a heavy growth of a Neisseria species. Only colonies morphologically consistent with neisseriae were present in the third streak zones of the blood agar and chocolate agar plates. Penicillin G, 2 x 106 U given intravenously every 4 h, was added. Forty-eight hours after adding the penicillin, the patient was afebrile and dramatically improved. The gentamicin and erythromycin were stopped and the penicillin was continued for 12 days. The chest roentgenogram cleared after 24 days. MATERIALS AND METHODS BACTEC Neisseria differentiation tests. The clinical isolate (UMC 2768) and a reference strain, N. cinerea ATCC 14685 (provided by the Centers for Disease Control), were tested in BACTEC Neisseria Differentiation Kits, using the BACTEC instrument (model no. 460). With this system, the test organism is suspended in a nutrient broth and inoculated into a set of three vials which contain `4C-labeled glucose, maltose, and fructose, respectively. After an incubation period of 3 h, the BACTEC instrument measures the amount of '4C-labeled C02 which is released as a result of utilization of the sugars. Identification procedures. At the Neisseria Reference Laboratory, Seattle, Wash., UMC 2768 was tested in modified oxidation-fermentation medium (7) for its ability to utilize glucose, maltose, fructose, lactose, and sucrose. Production of polysaccharide from sucrose, beta-lactamase production, and ability to reduce nitrate and nitrite were determined as previously described (2, 7). Auxotyping of strains was performed on chemically defined medium (NEDA) as described previously (6).

CASE REPORT A 25-year-old Haitian was found to have the acquired immune deficiency syndrome, based on the presence of central nervous system toxoplasmosis, chronic diarrhea due to Giardia lamblia, and reversal of the T-helper/suppressor cell ratio (OKT 4:8 = 0.1). Because of airway problems associated with declining neurological function, nasotracheal intubation with mechanical ventilation was initiated 2 days after hospitalization. Twenty-four days after institution of trimethoprim-sulfamethoxazole (14 days after addition of pyrimethamine), the patient still required mechanical ventilation due to continuing neurological dysfunction. His temperature rose to 102.6°F (39.3°C), the endotracheal secretions became purulent, and a chest roentgenogram showed a new infiltrate in the left lower lobe. Physical examination revealed the presence of bron*

Corresponding author. 1

2

J. CLIN. MICROBIOL.

BOYCE ET AL.

TABLE 1. BACTEC Neisseria Differentiation Kit results for N. cinerea UMC 2768 and ATCC 14685 BACTEC growth index Isolate Maltose

Fructose

Negative

Negative 10 4

Glucose

UMC 2768

ATCC 14685

650 687 315 571 644

5 2 3 9

12 21

194 160 240

2 0 2

9 il 4

Antimicrobial susceptibility tests. The susceptibility of UMC 2768 to penicillin, tetracycline, erythromycin, colistin, and spectinomycin was determined by using techniques described earlier (6, 8). RESULTS The gram-negative diplococcus recovered from the patient's endotracheal secretions grew on chocolate agar and on blood agar under increased C02 tension at 37°C and was oxidase positive. On chocolate agar the organism produced glistening gray colonies with smooth margins. There was no growth on Thayer-Martin medium at 37°C. The initial BACTEC Neisseria Differentiation Kit results yielded a positive growth index for glucose (650) and negative growth indices for maltose and fructose. Tests for ,-galactosidase activity and ,-lactamase production were negative. AIthough the results obtained with the BACTEC kit suggested that the organism might be N. gonorrhoeae, isolation of the organism from endotracheal secretions, growth of the organism on blood agar, and failure of the organism to grow on Thayer-Martin medium suggested that the isolate was not a gonococcus. The organism was sent to the Centers for Disease Control for identification. The organism failed to produce acid from glucose, mannitol, lactose, sucrose, or maltose in cystine-Trypticase (BBL Microbiology Systems) agar. It reduced nitrite but not nitrate, was DNase negative, and grew on nutrient agar at 25 and 35°C. There was no pigment production on Loeffler medium. The isolate was presumptively identified as probable N. cinerea by the Special Bacterial Pathogens Laboratory at the Centers for Disease Control. Since the organism failed to produce detectable acid from glucose when tested in cystine-Trypticase agar, repeat BACTEC Neisseria differentiation tests were performed to determine if the initial BACTEC results were reproducible. The organism consistently produced positive growth indices in vials containing [14C]glucose when tested on four other occasions (Table 1). N. cinerea ATCC 14685 also yielded positive growth indices when incubated in BACTEC vials containing [14C]glucose. The mean glucose growth index obtained with the clinical isolate (573.4) was significantly higher than the mean value obtained with the reference strain (198.0) (Student's t test, P < 0.01). The isolate was subsequently forwarded to the Neisseria Reference Laboratory for further characterization. The organism failed to produce detectable acid from glucose, maltose, fructose, sucrose, or lactose in modified oxidationfermentation medium and did not produce polysaccharide from sucrose. Nitrite was reduced, but nitrate was not reduced. Auxotyping studies revealed that the orga-

nism required proline, arginine, and cystine-cysteine (Pro-Arg-CC-) for growth. The isolate grew on tryptic soy and Mueller-Hinton agars. Antimicrobial susceptibility studies performed on the clinical isolate revealed the following MICs (micrograms per milliliter): penicillin (1.0), tetracycline (1.0), erythromycin (4.0), and spectinomycin (32). A disk diffusion test revealed that the organism was susceptible to colistin. On the basis of the above findings, the organism was identified as N. cinerea. DISCUSSION Initially, we suspected that the acute pneumonia was caused by N. meningitidis or B. catarrhalis. Representative colonies were subcultured to chocolate agar and to ThayerMartin medium. Although no growth was seen on ThayerMartin medium, we felt that the organism might be N. meningitidis, since some meningococci do not grow well on commercially prepared Thayer-Martin medium. BACTEC Neisseria differentiation tests were performed on the isolate in an attempt to identify the Neisseria sp. and to determine the possible need for antimicrobial prophylaxis for exposed hospital personnel. A positive BACTEC growth index for glucose, with negative indices for maltose and fructose, suggested that UMC 2768 might be N. gonorrhoeae, a "late maltose-fermenting" group C N. meningitidis, or a maltose-negative N. meningitidis (3, 5). However, UMC 2768 was differentiated from N. gonorrhoeae and N. meningitidis on the basis of the following characteristics: failure to produce detectable acid from glucose or maltose in modified oxidation-fermentation medium, growth on tryptic soy and Mueller-Hinton agars, susceptibility to colisitin, and requirement for proline, arginine, and cystine-cysteine for growth on NEDA medium (8). N. cinerea may be confused with other asaccharolytic gram-negative diplococci such as N. flavescens or B. catarrhalis, especially when isolates recovered from the respiratory tract are tested only in cystine-Trypticase agar or in modified oxidation-fermentation medium. However, B. catarrhalis reduces nitrate, whereas N. cinerea does not (8). N. flavescens is capable of producing polysaccharide from sucrose, whereas N. cinerea is not. The ability of UMC 2768 to metabolize ['4C]glucose in the BACTEC system, without producing acid from glucose in cystine-Trypticase agar or in modified oxidation-fermentation medium, remains an enigma at this point. We have tested only one other confirmed N. cinerea isolate (ATCC 14685), and it also yielded positive glucose indices in the BACTEC system. Additional N. cinerea isolates are being tested to see if they react similarly. If this trait is common among N. cinerea isolates, the potential for misidentification of N. cinerea as N. gonorrhoeae may occur in laboratories that rely on the BACTEC system for identification of path-

ogenic neisseriae. ACKNOWLEDGMENT We thank Robert E. Weaver for performing the initial biochemical reactions which led to the correct identification of UMC 2768. LITERATURE CITED 1. Berger, U. 1963. Die anspruchslosen Neisserien. Ergeb. Mikrobiol. Immunitaetsforsch. Exp. Ther. 36:97-167. 2. Berger, U., and E. Paepcke. 1962. Untersuchengen uber die asaccharolytischen Neisserien des menschlichen Nasopharynx. Z. Hyg. 148:269-281. 3. Granato, P. A., R. Howard, B. Wilkinson, and J. Laser. 1980. Meningitis caused by maltose-negative variant of Neisseria

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meningitidis. J. Clin. Microbiol. 11:270-273. 4. Henriksen, S. D. 1946. Isolation of atypical strains of Neisseria catarrhalis from the genito-urinary tract. Acta Derm. Venereol. 26:506-514. 5. Johnston Laboratories. 1980. BACTEC Neisseria Differentiation Kit, model ND-1 package insert. Johnston Laboratories, Cockeysville, Md. 6. Knapp, J. S., and K. K. Holmes. 1975. Disseminated gonococcal infections caused by Neisseria gonorrhoeae with unique nutritional requirements. J. Infect. Dis. 132:204-208. 7. Knapp, J. S., and K. K. Holmes. 1983. Modified oxidation-fermentation medium for detection of acid production from carbohydrates by Neisseria spp. and Branhamella catarrhalis. J. Clin. Microbiol. 18:56-62. 8. Knapp, J. S., P. A. Totten, M. H. Mulks, and B. H. Minshew.

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3

1984. Characterization of Neisseria cinerea, a nonpathogenic species isolated on Martin-Lewis medium selective for pathogenic Neisseria spp. J. Clin. Microbiol. 19:63-67. Murray, E. G. D. 1939. Genus I. Neisseria Trevisan, p. 278-288. In D. H. Bergey, R. S. Breed, E. G. D. Murray, and A. P. Hitchins (ed.), Bergey's manual of determinative bacteriology, 5th ed. The Williams & Wilkins Co., Baltimore. Reyn, A. 1948. Atypical gonococcal strains. Acta Derm. Venereol. 28:381-386. von Lingelsheim, W. 1906. Die bakteriologischen Arbeiten der Kgl hygienischen Station zu Beuthen O. Sch. wahrend der Genickstarreepidemie in Oberschlesien im Winter 1904/05. Klin. Jahrb. 15:373-489. Wax, L. 1950. The identity of Neisseria other than the gonococcus from the genito-urinary tract. J. Vener. Dis. Inf. 31:208.