Meningitis may be caused by several distinct species of bacteria, viruses and protozoa. The initial clinical diagnosis is usually confirmed by chemical and cytological examination of the C.S.F. and the causal pathogen identified by bacteriological and virological techniques.  A case report of a 5-year old boy who was admitted to St. Luke's Hospital Malta with a history of mild frontal headaches for the previous two days is presented. A diagnosis of meningitis was made and a lumbar puncture performed. The fluid was clear and the C.S.F. pressure was found to be within normal limits. In the case under discussion, one can argue that the cause of the meningitis may have been the Flavobacterium which was cultured from the blood but for some reason not from the C.S.F. As an additional note it may be mentioned that this child comes from a family of 4 children, including himself. One brother died soon after birth. The other two siblings were both admitted to hospital in with meningeal signs. Although a definite correlation between these three cases of 'meningeal involvement' cannot be proved, it is peculiar that 3 siblings in the same family should be affected by a common disease within a span of 6 months.peer-reviewe

Pepper, Rosemary

Portelli, Alfred V.

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122 A FLAVOBACTERI UM CULTURED FROM THE BLOOD OF A PATIENT WITH MENINGITIS A.V. Portelli M.D., M.Sc. (B'ham), Dip. Bact. (Lond). and R. Pepper B.Sc. (Reading), M.Sc. (Cantab). Depa1'Dment of BaCtterioLogy, St. Luke's Hospitab Introduction Meningitis may be caused by several distinct species of bacteria, viruses and protozoa. The initial clinical diagnosis is usually confirmed by chemical and cytolo-gical examination of the C.S.F. and the causal pathogen identified by bacteriolo-gical and virological techniques. Bacterial meningitis is often caused by Neisseria meningWd;ils, HaemophvLus inlfLuenzae and various streptococcal species. Less common causative organisms belong to the Flavo-bacterium group. These are Gram-nega-tive, slender, non-motile bacilli having restricted biochemical activities and are often resistant Ito several antibiotics, inc:ud-ing penicillin. Some strains produce a pro-nounced yellow pigment. The earliest description of this organ-ism was given by Franklands (1889) who isolated it from deep wells in Kent. How-ever, owing to incompleteness in the origi-nal description, one cannot be certain of its exact identity. Thereafter, several authors recorded the isolation of bacteria with characteristics s'imilar to those des-cribed above and in circumstances sug-gesting that they were pathogenic for man. More recently, outbreaks of menin-gitis in new born infants caused by Flavo-baCteria were recorded by Vandepitie, Beeckmans and Buttiaux (1958) in the Congo and by Brody, Moore and King (1958) and George, Cochran and Wheeler (1961) in the United States. Cabrera and Davis (1961) put forwalrd evidence to sug-gest that Flavobacteria usually lead a sap-rophytic existence but may also colonize the human nose. The Flavobacterium genus contains 2 groups designated group lIa, named navobaoterittm meningosepticum and group lIb tentatively cla;ssified as FLavo-bacterium sp. (King, 1959). Strains in group lIa are often associated with infant meningitis and, unlike members of group lIb which produce a pronounced yellow pigment, show little or no pigmentation. These 2 groups although closely related show some differences in other biochemi-cal reactions which enable laboratory workers to distinguish them (Tatum, 1970), Clinic ail features and laboratory investigations A 5-year old boy was admitted to St. Luke's hospital on the 5th November, 1973 with a history of mild frontal head-aches for the previous 2 days. The pain later extended to the occipital region and to ,the neck. He vomited once, a few hours prior to admission to hospital. On examination, his general condi-tion was fair. The temperature was 101°F and ,the pulse rate was 140/min. Neck rigidity was present and Kernig's sign was positive. No other abnormalities were detected.' A diagnosis of meningitis was made ,and a lumbar punoture performed. The fluid was clear and the C.S.F. pressure was found to be within normal limits. The chemical and cytological examination gave the following result:-Physical findings: fluid clear and co-lourless. Globulin test: positive. Proteins: 40 mg/100 ml. Glucose: 44 mg/100 ml. Cells: 30/cu. mm. the great majority of which were polymorphs. Bacteriological examination of the sample was negative both on direot examination and on subse-quent culture. Other investigations included Hb es-timation, WBC counts and blood urea levels. All were found ,to be within normal limits. The patient was put on Penicillin 2 mega units 8 hourly, Chloramphenicol 125 mg 6 hourly and Sulphadiazine 125 mg 6 hour:y. He recovered fairly rapidly and became afebrfe on the 3rd day after admission. The CNS signs subsided about this time. He continued to make good pro-gress and was discharged from hospital on the 8th day after admission. Blood culture was a1so performed on admission. This consisted in taking 5 ml of blood under aseptic conditions from a suitable vein and inocu -ating it into a Cas-taneda bottle. This is simply a m2dical flat bottle of 100 ml. capacity in which is pre-pared a doub~e (solid/liquid) medium. 'fryptose agar constitutes the solid phase and covers one broad side of the bOttle to a depth of about 5 mm. The liquid phase is made up of 35 cc tryptose phosphate broth with an anticoagulant which is ad-ded to each bottle after the solid phase has set firmly. The metal cap which has a rub-ber lining on the inside is perforated by a central hole of about 4 mm in diameter. Blood taken from a patient with a syringe is inoculated directly into the liquid phase of the medium without opening the bottle. The inoculated blood culture bottle was incubated in an upright position at 37°C and examined daily. The solid medium was sub cultured from ,the liquid phase on alter-nate days by gently shaking and tilting th2 bottle so that the liquid medium just covered the solid phase. On the third day after incubation, a yellowish confluent growth was detected. A Gram stain showed a small rod about 1.5p. x 0.4p. which was Gram-negative and non-acid-fast. Subcul-tures from the original blood culture bottle were made onto appropriate media and fur-ther tests to enable identification of this bacillus were carried out. 123 The bacillus was found to be non-motre. It grew well on tryptose agar after 24 hours at 37°C forming smooth circular colonies which showed pronounced yellow pigmentation wHh a faintly greenish iri-descence. The plgment was insoluble in water. No haemolysis was observed on blood agar. Growth on MacConkey's agar was not present at first but occurred later after some subcultures had been done. No growth at all was seen on SS agar. The catalase and oxidase tests were positive. Citrate utilization (Koser's), M.R. and V.P. tests were negative. Urea (Christensen's agar) was not hydrolysed. Indole produc-tion was very weak. Gelatin was hydrolysed by the third day. Growth was maximal at 37°C with no growth occurring at all at 5°C. Acid production was negative in liquid carbohydrate nutrient media employing several sugars. To test whether the attack on sugars was of the oxidative or fe1'-mentative type, the oxidation-fermen-tation (0 - F) test of Hugh and Leifson (1953) was employed using glucose and suc-rose. Acid production from glucose was observed in the open tube by the 3rd day; the result was doubtful in the sea!ed tube. The test was negative for sucrose in both tubes. On testing the organism for antibiotic sensitivity it was found that it was sensitive to chloramphenicol, moderately sensitive to trimethoprim-sulphamethoxazo~e and gen-tamycin and resistant to penicillin, strepto-mycin, amoxil, tetracycline, polymyxin, and penbrltin. The cultural characters and biochemical reactions are summarised in Table 1 and compared with King's groups Ha and Hb. With these findings we tentatively placed the isolate in the Flavobacterium genus and possibly belonging to King's group lIb. Slide and tube agglutination tests with the patient's serum against a suspension of the cultured organism were negative. Nasal and throat swabs taken later from the patient and his 10-year old sister were nega-tive for Flavobacterium. Discussion In a blood culture examination we al-ways presume that a growth obtained after incubation is a potential pathogen which is 124 Chal'acters FLavobacterium F~avobacterium sp. Isoiate meningosepticum referred to Group lIa Group !Ib in text Gram stain negative negative negative Motility non-motile non-motile non-motile Pigment (yellow) none or very slight pronounced pronounced Growth (In usually positive after usually postive positive after MacConkey's agar several subcultures after several several subcultures subcultures Growth on SS agar none nCfne none Catalase positive positive positive Oxidase positive positive positive O-F medium acid acid acid unsealed tubes some some by the (glucose) delayed delayed 3rd day Indole weakly positivc weakly positive v. weak positive U rea hydrolysis negative negative negative Citrate utilization negative negative negative Gelatin liquefaction positive positive positive Pathogenicity associated with infant recovered mainly recovered from meningitis from adults 5-yr old ched Table 1. Comparison of some characters of isolate referred to in the text with those of King's groups lIa and lIb. causing a bacteraemia or septicaemia in the case concerned. Although blood is taken under strict aseptic precautions, contamina-tions do at times occur. This was shown by Cameron, Rae and Murphy (1931) who made blood cultures from 100 healthy individuals and obtained positive results in 18 cases. Of the positive blood cultures, 12 were shown to be due to large spore-bearing bacilli and were regarded as contaminants. Some, at least, of the remaining 6 positive blood cuI-tures which included isolations of Staph. au-reus ap.d of diphtheroids were also dis missed as contaminants. Some cases are clear cut in the sense that it can be safely stated that the organism cultivated from the blood is causing the under:ylng disease in the patient. Thus, the isolation of SalmoneUa typhi or BruceuUz meLvtensis from the blood, especially if relevant clini-cal signs and symptoms are present, is ir-refutable evidence that the patient is suf-fering from the particular disease. At other times it is difficult to state whether an iso-late is the cause of a bacteraemia or sep-ticaemia or whether it is a contaminant picked up, say, from the patient's skin dur-ing the blood taking procedure. Points to help decide the issue include correlation of a particular positive blood culture with the clinical picture, repeated b~ood culture examinations and the performance of other supporting bacteriological itests. In the case of culturing Staph_ aJhbus from the blood, to mention one example, a diagnosis of an in-fection with this organism ShOUld never be made from the result of only one test. A bacteraemia due to this organism can be safely assumed following rep3ated isolation. In the case under discussion, one can argue that the cause of the meningitis may have been the Flavobacterium which was cultured from the blood but for some reason not from the C.S.F. In favour of th:s state-ment is the fact that this organism is now recognised as being one of the causes of meningitis and culturing it from the b~ood is at least evidence of its presence in the tissues. It is not inconceivable that it may have found its way also into the C.S.F_ Other possibilities include the proposition that the cause of the meningitis may have be3n some other type of organism and the posi-tive blood culture. was a separate, unrelated event. This however seems unlikely as it is difficult, although not impossible, to imagine a double pathology of ths nature_ Lastly, the Flavobacterium may have been pick>ed up as a contaminant from the . patient's skin or from some other source during the blood culture procedure. In favour of this argument is the mild nature of the disease in this case (although this may have been due to early diagnosis and prompt successful treatment), the absence of leucocytosis and the fact that the patient's' serum did not contain antibodies against .the organism supposedly cultured from his blood. A~ an additional note it may be men-tioned that this child comes: from a family of 4 children. including himse'f. One brother died soon after birth. The other 2 125' siblings were both admitted to hosp:tal in May 1973 with meningeal signs. One, a brother of 11 years was diagnosed as 'Fever' accomp:mied by meningeal irritation but no C.S.F. or b:ood culture examinations were carried out. The illness was mild and the patient was discharged from hospital after a few days stay. The other, a 10-year old sister was admitted to hospital with severe meningitis having a total of 13,300 ce:ls/ cu. mm in the C.S.F., the majority being polymorphs. A coagulase negatIve staphylo-coccus was cultured from the spinal fluid but this was thought to be a contaminant. The patient eventually recovered. Although a definite correlation between these three cases of 'meningeal involvement' cannot be proved, it is peculiar that 3 sib~ings in the same family should be affected by a com-mon disease within a span of 6 months. Acknowledgements Our thanks are due to Dr. E. Agius, the Senior Bacteriologist iin our· Department, for his helpful comments and encourage-ment, to Dr. T. Agius Ferrante for permis-sion to pablish this case and to Dr. E.A. Cachia for clinical information regarding the sib-ings of the patient. References BRODY, J.A., MOORE, H. and KING, E.O. (1958). Amer. J. Dis. Child., 96, 1. CABRERA, H.A. and DAVIS, G.H. (1961). Amer. J. Dis. Child., 101, 28!1. CAMERON, G.C., RAE, C.A. and MURPHY, G.N. (1931). Canad. M.A.J., 25, 131. FRANKLAND, G.C. and FRANKLAND, P.F. (1889). Z. HYg. Infekt Kr., 6, 373. GEORGE, R.M., COCHRAN, C.P. and WHEELER, W.E. (1961). Amer. J. Dis. Child., 101,296. HUGH, R. and LEIFSON, E. (1953). J. Bact., 66, 24· > KING, E.O. (1959). Amer. J. Clin. Path., 31, 241. TJ\.TUM, H.W. (1970). Manual of Clinical Micro-biology p. 197. American Society for Micro-biology Bethesda, Md. V ANDEPITTE, J., BEEC:KJIt".ANS, C. and BUTTIAUX, R. (1958). A~. Soc. beIge Med. trop., 38, 563. 

A flavobacterium cultured from the blood of a patient with meningitis

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A flavobacterium cultured from the blood of a patient with meningitis

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