J Clin Pathol 1983;36:1097-1 1 01

Anaerobiospirillum species isolated from humanswith diarrhoeaH MALNICK, MEM THOMAS,* H LOTAY,* M ROBBINS

From the National Collection of Type Cultures and Epidemiological Research Laboratory, Central PublicHealth Laboratory, London NW9 5HT, and the *Department of Clinical Microbiology, University CollegeHospital, London WCJ E 6AU

SUMMARY Flagellated anaerobic motile spiral bacteria were isolated from the faeces of twopatients with diarrhoea. They were recovered by the microaerophilic culture method used todetect campylobacters but demanded anaerobic conditions for subculture. Electron microscopyand other investigations showed them to be closely related to Anaerobiospirillum succini-producens first described in beagle dogs and subsequently in three humans with bacteraemia.

Anaerobic spiral bacteria in the human bowel havenot often been reported. When found, suchorganisms have been of two types, spirochaetes withaxial fibrils'-7 and non-spirochaetes without visibleaxial fibrils.8 We here report the isolation of relat-ively anaerobic flagellated spiral bacteria from thefaeces of two patients with diarrhoea.

Material and methods

The organisms were isolated during the routinediagnostic examination of faecal specimens. The firstpatient (A30) was a 41-year-old man who hadrecurrent pain and loose stools, the second (A142)was a child who attended a local Day Nursery.Rotavirus, Shigella, Salmonella and Campylobacterspecies were not found in either patient. Faecesfrom 34 child contacts of A142 were examined, butthe spiral organism was found in this one case only.Six other children were excreting Shigella sonnei.The two spiral organisms were designated A30

and A142 respectively.

CULTURE METHODSThe methods in routine use for the recognition ofintestinal pathogens included a selective medium forcampylobacters containing vancomycin, polymyxinand trimethoprim9 which was incubated for 48 h at43°C employing a BBL Campylobacter ("Cam-pypak") sachet in a jar to secure a microaerophilicatmosphere.Both strains of spiral bacterium were initially

isolated by this method, but anaerobic incubationAccepted for publication 25 May 1983

was required for regular successful subculture.Oxygen and temperature tolerance were inves-tigated in parallel on 5% horse blood agar, choco-late agar, lysed blood agar and buffered charcoalyeast extract agar (BCY)'0 using an evacuation sys-tem in an anaerobic jar, incubated at a range oftemperatures between 30°C and 45°C. Ninety percent hydrogen and 10% carbon dioxide gas wereutilised as a basic atmosphere to obtain a com-parison between oxygen concentrations of 5%, 4%and 3*5% and strict anaerobiosis.

FERMENTATION AND BIOCHEMICAL TESTSCell suspensions in distilled water were made from24-hour anaerobic cultures on blood agar and usedfor inoculation. The methods of Cowan and Steel"were used with the following modifications; skim-med milk powder was used for testing casein hyd-rolysis; sodium nitrate for nitrate reduction and0*4% starch for starch hydrolysis. The blue catalasetest,'2 which traps bubbles, was compared with thestandard method. The basal medium for testing acidproduction from carbohydrates and glucose fermen-tation products was peptone yeast broth.'3 The endproducts of glucose fermentation were analysed bygas liquid chromatography on a Pye Unicam Series204 Chromatograph.

ANTIBIOTIC SENSITIVITYThe disc method was used on blood agar and BCYincubated anaerobically at 37°C and 43°C. A strainof Campylobacter jejuni freshly isolated from ablood culture was tested in parallel on mediaincubated microaeropliilically.

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CELL MORPHOLOGYGram-stained impression preparations were

examined after 24 and 48 h culture on 5% horseblood agar and BCY in microaerophilic andanaerobic atmospheres at 37°C and at 43°C. Forelectron microscopy (EM), cultures on BCYmedium incubated anaerobically at 37°C for 24 and48 h were scraped and suspended in 2% formal-dehyde solution. The bacterial cells were stainedwith 1% phosphotungstic acid and then examinedunder an AEI EM6B electron microscope. Thewavelength and amplitude of the spirals seen were

measured on EM photographs and compared withthose recorded for typical Anaerobiospirillum suc-

ciniproducens'4 and Campylobacter jejunil5 strains(Table 1).

Results

MORPHOLOGYThe initial cultures A30 and A142 were obtained oncampylobacter selective medium incubated micro-aerophilically at 43°C for 48 h. They resembledCampylobacter jejuni in colonial and microscopic

Malnick, Thomas, Lotay, Robbins

appearance but were oxidase-negative and weredifficult and often impossible to subculture micro-aerophilically. However they grew well after 24 h inan anaerobic atmosphere on 5% horse blood agar,chocolate agar, lysed blood agar and BCY medium,but not on nutrient agar. The temperature toleranceextended from 32°C to 430C. Colonies on bloodagar after 24 hours at 370C were 0*5 to 1 mm indiameter, translucent and non-haemolytic, withsome delicate spreading or swarming peripheralgrowth which was more marked at 43°C than at370C (Fig. 1). Impression films showed Gram-negative spiral organisms 3-5 am long and 0 5 umwide (Fig. 2), after 24 hours incubation but at 48hours the cells had elongated up to 34 ,um with as

many as 20 regularly spaced spirals (Fig. 3). Oldercultures became progressively more fragmented andcoccoid. Dark field microscopy revealed spiralmotile cells which revolved around their long axes.

Electron microscopy showed these cells to havebipolar tufts of multiple flagellae (Figs. 4 and 5).Organism A30 had a spiral wavelength of 1.4-2 ,smwith an amplitude of 0O23-0-35 ,um, while A142 hada wavelength of 1-7-2.2 um and an amplitude of0.2-0-65 um (Table 1).

Table 1 Characteristics ofspiral organisms A30 and A142 compared with those ofAnaerobiospirillumsucciniproducens'4 and Campylobacter jejuniI-

A30 A142 A succiniproducens C jejuni

Acid from:Fructose + - +Glucose + + +Inositol - -

Lactose - - +Maltose - - vMannitol - -

Raffinose - - +Salicin - -

Sorbitol - -

Sucrose - - +Trehalose + - v

Casein - - NTOxidase +Catalase - - - +Blue catalase test trace trace trace +Glucose fermentation:

acetic & succinic acidas major end products + + +

Subculture growth:anaerobic + + +aerobic - -

3-5-5% 02 - - - +Lecithinase - - vMethyl red - - NTNitrate reduction - - - +ONPG - - NTPhosphatase - - NT NTStarch hydrolysis - - v -Temperature:Maidmum 430C 43°C 40°C approx 430CMinimum 320C 320C 25°C approx 3rC approx

Urea hydrolysis -

Spiral wavelength (gtm) 1-4-2 1-7-2-2 1.31-7 0-9-1.3Amplitude (Jm) 0-23-0-35 0*2-0&65 09-1*1 0-35-0.6

v = variable, NT = not tested.

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Fig. 1 Feathery swarming growth ofAnaerobiospirillumon surface ofblood agar after 48 hours anaerobicincubation x2.

BIOCHEMICAL AND FERMENTATION TESTSThe results are summarised in Table 1 whichincludes a comparison with some reactions charac-teristic of type strains of Anaerobiospirillum suc-ciniproducens and of Campylobacter jejuni. Oxidasetests were negative. Conventional catalase testswere also negative, but a trace reaction was obtainedconsistently with the more sensitive blue peroxide

Fig. 2 Impression film ofAnaerobiospirillum culturedanaerobically on blood agar for 48 hours, stained Gramx1600.

Fig. 3 Electron micrograph ofAnaerobiospirillumcultured on BCY for 48 hours, showing many spiralsx2250.

catalase test'2 with which the type strain of A suc-ciniproducens also gave a trace reaction. Gas liquidchromatography detected acetic and succinic acidsas major end products of glucose fermentation byboth A30 and A142, as by the type species A suc-ciniproducens; but not by C jejuni.The two spiral organisms A30 and A142 resemb-

led each other and A succiniproducens in culturalrequirements and morphology but differed slightlyin biochemical tests. They differed culturally, mor-phologically and biochemically from campylobac-ters.

Fig. 4 Electron micrograph ofAnaerobiospirillumcultured on BCY for 24 hours, showing bipolar tufts offlagella x13 500.

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Fig. 5 Electron micrograph ofAnaerobiospirillumcultured on BCY, showing flagella tuft at highermagnification x29 250.

ANTIMICROBIAL SENSITIVITYThe results of disc sensitivity tests are shown inTable 2. They were the same on BCY and bloodagar plates at 37°C and 43°C. Apart from a differ-ence in the degree of resistance to gentamicin, A30and A142 were alike. They were, unlike C jejuni,more sensitive to penicillin than to erythromycin.Although sensitive to the standard 300 unitpolymyxin disc they grew on a campylobacterselective medium9 which contained polymyxin 2x5units per ml.

Discussion

There have not been many reports of human intesti-nal infection by elongated spiral bacteria. In recent

Malnick, Thomas, Lotay, Robbins

descriptions the diagnosis has been achieved by his-tological examination of biopsy material and byelectron microscopy.'-8 16 Most of these reportsdescribe bacteria with axial filaments, confirmingthat they belong to the Family Spirochaetaceae.Takeuchi et al3 17 however, observed an organismwith single bipolar flagella in colonic epitheliumfrom humans and from rhesus monkeys, but did notindicate whether it had been cultured. Two reportsconcern long spiral bacteria which were successfullycultured; one of these showed neither axial fibrilsnor flagella8 and the other was not examined by elec-tron microscopy.'8 In 1976 Davis et al'4 reported theisolation of an Anaerobiospirillum species from themouth and faeces of beagle dogs and proposed thetype species A succiniproducens. This organism hassince been recovered from blood cultures in threefebrile adult human patients.'920 Like A succini-producens, the two anaerobic spiral organisms wedescribe have bipolar tufts of flagella, but they dif-fer slightly from the type strain and from eachother in their pattern of carbohydrate fermentation.On the basis of tests carried out so far we concludethat A30 and A142 are Anaerobiospirillum species,but not necessarily A succiniproducens. Althoughthey were isolated from the faeces of patients suf-fering from diarrhoea there is insufficient evidenceon which to ascribe pathogenicity. It may howeverprove worthwhile to seek such bacteria in future.Although essentially anaerobic and intolerant ofoxygen on subculture, both A30 and A142 wereinitially isolated microaerophilically on a cam-pylobacter selective medium incubated at 43°C for48 h and the strains were moderately sensitive tometronidazole. At that stage the colonial form andthe appearance of Gram-stained bacteria closelyresembled those of a campylobacter although some

Table 2 Sensitivity ofA30 and A142 to antimicrobial agents

Antimicrobial Disc content (IU/pg) A30 A142 Campylobacter jejunit

Penicillin 2 IU S S R*Polymyxin 300 IU S S R

Ampicillin 10 S S SCephalexin 30 S S RChloramphenicol 10 S S SErythromyan 10 M M SFusidic acid 10 R R RGentamicin 10 S M SMetronidazole 5 M M RNalidixic acid 30 M M SNitrofurantoin 50 S S SSulphafurazole 100 S S STetracycline 10 S S S*Tnmethopnm 1-25 R R R*Vancomycin 35 R R R

S = sensitive; M = moderately sensitive; R = resistant.*Component of the campylobacter selective medium used for isolation.tStrain from a blood culture.

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Table 3 Typical characteristics ofnon-aerobic spiral bacteria2' 22

Flagella Growth at Oxidase Catalase250'C 3S°C 40°C (conventional)

MicroaerophilsCampylobacter spp 1 polar v + v + +Spirillum volutans bipolar tuft + + -+ +AnaerobesAnaerobiospirillum spp bipolar tuft v + +Desulfovibrio spp polar tuft + + +Wolinella spp polar tuft - +Selenomonas spp lateral v + vButyrivibrio spp 1 polar - + +Succinivibrio spp 1 polar - + --Anaerobic spirochaetes axial only v + -v

v = variable.

spiral cells were very long.Campylobacterjejuni isolated from faeces at 43°C

are characteristically oxidase and catalase positiveby conventional methods and most strains are fullysensitive to erythromycin, so the negative results weobtained with these three tests indicated the needfor further study. Biochemical tests were not foundvery helpful until an identification to Family levelwas achieved. At present, as is seen in Table 3, elec-tron microscopy is the best way to discriminate be-tween the known groups of spiral bacteria.

We are grateful to Dr L R Hill, National Collectionof Type Cultures for his advice and thank AA Porterand C Paddon of the PHLS Virus Reference Lab-oratory for the electron micrographs, J Gibson ofthe Photographic Department for the micro-photographs and F Scott for typing the manuscript.

Requests

Mr H Malnick, National Collection of Type Cul-tures, Central Public Health Laboratory, Colindale,London NW9 5HT would be grateful to receivestrains or information about similar spiral organismsisolated elsewhere

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Requests for reprints to: Dr Mair EM Thomas, CentralPublic Health Laboratory, Colindale Avenue, LondonNW9 5HT, England.

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