Gram-positive anaerobic streptococci (GPAC)

David Murdoch, MA, MD, MRCPath Department of Medical Microbiology, Southmead Hospital NHS Trust,

Bristol BS I 0 5NB, UK

Introduction

I l11erest in anaerobes has passed through several phases. For many years, it focussed on clostridia because of their importance in wound infections. Later, Gram-negative anaerobic rods (GNAR) attracted much attention becHuse of the pathogenicity of organisms such as Boclemides ji'ogilis. M ore recellt ly, ClostridiulII dUfici/e has been intensively studied because of i ts importance in antibiotic-assoc iated diarrhoea. In contrast, peplOstreptococci, or Gram-positi ve anaerob ic cocci (GPAC), have been relati vely neglected, I although they are often cultured from signilicant in fections. 1.2

Several factors have contributed to this lack of il1lerest:

• the c lassi fication is very unsatisfacLOry ;3 several poorly defined species, notably I~ asacc/wrolyliclfs and P prevolii , ~Ire no\-v recognised to be genetically hCICrogclleolls; 1.4

• the identific.ation methods which work well for clostr idia and GN AR are of l ittle use for GPAC bu t are still used: 1.5.6

• GPAC are often cultured from mi xed infections, part icularl y of Illllcosal surfaces, in which their significance can be questioned. I

The result has been a lack or c lini ca l interes t, di scouraging laboratory research and perpetuating a cycle of neglec t (Figu re I ).

Definition

The study or GPA C has suffered from a proliferati on of synonyms; mi snomers such as 'anaerobic strepLOcoccUS' are still lIsed to describe them. The term GPAC is preferable because it is useful in the routine diagnostic laboratory;1 it i s~ a term of convenience wi th no taxonomic implications, giving a broad morphological description of organi sms isolated under speci fi ed atmospheric condit ions. Watt and Jack7 defined anaerobic cocci as 'cocci that grow well under sati sfactory conditions of anaerob iosis and do not grow on suitable solid media in 10% CO., in ,Iir e ven after incubation for 7 chlys .If

37°C. ' This va lua-ble def inition excludes micro-aeroph ilic species but includes genera of extremely oxygen-sensit ive

organisms sllch as Rlllllil/oCOCCIIS, which are rarely isolated from cl inical specimens and wi ll not be discussed further.

The species of most cl inical impol1ance are PeprosrrepfOcocclfS I/Jag l/lls, PeproslreplococClIs micros, Peprosirepiococci/s ClSClCC/Wro/yficlls and Peplosll ep lococCIIS ClIWelVbitls; these four spec ies accounted for. over 75% of all cli nical isolates ill a recent survey.8

The v icious circle

~Little interest~

Can't be Not important identified /

~won't bother ~ Figure I. 'The vicious cycle' - why GPAC have been litt le studied

Classification

For 50 years, 1110st GPAC or medical impol1ance were separated into two genera, PeplOcocclIS and Peproslreplococclfs , ori ginally on the basis of the Gram-stained morphology, and laler, poorl y defined metabo lic differences . With the applicati on of nucleic acid-based techniques in the earl y 1980s, most spec ies were reclassifi ed in the genus PeploslreplococCIfS . M ore recentl y, 16S rRN A sequencing data have revealed that PeplosrreplococClfS is a phy logenetically heterogeneous taxon;3.4 it wi ll probably be

Also in this issue (page 5):

Vibrios 011 the half-shell Professor J. Glenn Morris Universily of Maryland, USA

Growt h of Vcholeroe {rig ht} and V vufni(icus (l eft) on thi osulphate citmte bile-salts sucrose (TCBS) selective medium.

I I

ClI/tlire Vol 20 No I .............................. ..

Table I. Recognised species of Pe ptost rc ptocOCClis with possible revised classification based on 16S rRNA sequence analysis,I.3."

Genus Species Terminal VFA

PCplOSI rcptococC liS (lIwerobiw; (T) IC (IV)

Genus I prevorii B Ie /rodills B IIycl!vgelllllis B /cU.:IO/yl ;CI/,\' B vag illa/is B 'octavius' C

Genus 2 asacc/wro/yficils B illdoliC/ls B

Genus 3 magI/lis A

Genus 4 micros A

Gellus 5 bome.l'oe A(B)

Gellus 6 lacrill/o/is B

Genus 7 '/wrei' B

Gellus 8 ' I I/Or/CIIS IV

?Ru lllinococc lis prodllC/IIS A

?Eubactcrium iteliolrillredllcells A

VFA, vo latil e fatty acid: A. aCClalC; B, butyrate: IV. isovalcratc: IC. isocaproalc: C. n-caproa\c: VFAs in brackets refer \0 rarely fOfmed terminal YFAs. (T). type spec ies of gellUS. Species in bold arc the clinically most important: species ill pare ntheses are recently descri bed.

spli t into at least 8 genera.IA The results will initially be confusing (Table 1): the type spec ies of the genus, P. al/aeJVbills, is more closely related to some clostridia than to any other species of PeprosrreprococclfS, so, by the rules, all the other spec ies wi ll have to be assigned to other (new) genera! I However, reclassification wi ll lead to a much stronger correlalion between taxon and biology in areas such as biochemistry, ecology and antibiotic sensitivity pallerns.

Laboratory isolation

Clinical specimens must be carefu lly collected and rapidly transported to the laboratory; if there is any delay, they must be stored in a good transport meclium.9 Specimens of pus or liss lle are 1110st likely to yield GPAC; dry swabs are of little use. GPAC require an anaerobic atmosphere containing CO2 in which to multiply but it is likely that many strains from clinical specimens can survive for some time in air. Most GPAC are fast idi ous, growing best on enriched, blood­containin g media such as Fastidious Anaerobe Agar (FAA),I. IO forming colonies of' diameter Iml11 after incubation for 48 hours; however, it is recommended that cultures fro m 'valuabl e' specimens be incubated for at least 5 days as otherwise many isolates wi ll be mi ssed (Figu re 2). The lack or <l sati sfactory selecti ve medium is a major limitation as GPAC are often co-cultured with organisms such as co'liforms, which grow so llluch faster that the GPAC ...... are not detected. I The development of selective media and investigations of individual species' growth requirements and aero tolerance would be valuable; it is very unfortunate that these aspects of their biology have been so lill ie studied.

Laboratory identification

Potential isolates of GPAC must first be distinguished from l1licro-aerophi l ic organisms such as ' mi Il eri ' group

2

Figure 2. Colonial morphology of typical stmin of P. 1I/(I&"I1S after 5 days' incub'l\ion.

streptococci, Staphylococcus sacclial'O/ylicw; and Gell1ella 11Iorbillorlllll, which are not strict anaerobes but often do not grow aerobically on primary culture. A chea p and reliable technique is to place a metronidazole 5~g di sc on the edge of the plate at one end of the primary inoculum, well away from secondary streaking; micro-aerophi les are resistant to metronidazole but almost all GPAC arc sensit ive. 1 Another possible pitfa ll is the tendency of some species, nOlably P. asacc/wroly licJ('\", to appear Gram-negati ve; checking with a vancomycin 5~ g disc prevents misidentifica tion as GPAC are sensitive to vancomyci n.

How much further ident ification is taken depends on the specimen and the resources available; identitica tion to the species level should be considered when GPAC are isolated in pure culture or from ii11ponant specimens such as deep organ abscesses, as this informat ion can guide choice of antibiotic. I Standard manuals sti ll base identification on carbohydrate fermentat ion reac tions and detection of volatile fatty acids (VFAs) using gas-liquid chromatography (GLC)5.6 However, GLC is of l imited use except for P. allCierobilts: few rout ine laboratories can afford the capita l costs or human resources that OLC requires. As few GPAC ferment carbohydrates, identification by standard techniques is usual ly based on a series of negative tests, wh ich probably explain s why few routi ne laboratories previollsly attcmpted fu ll identifi cati on. 1 Identification methods shou ld be appropriate to the metabolism of the organisms concerned. Most GPAC (Irc' proteolytic; wi th the developmen t of preformed enzyme profile (PEP) kits sllch as RAPID lD 32A,9.11 which incorporate a range of proteolytic and saccharolytic enzy mes, rou tine laboratories can now identi fy Illost';solmes from clinical specimens, includi ng those of P lI/agl/lIs, I~ lIIiCIVS and I~ {/I/{/erobius (Figure 3). 1.8 Several peptidase tests di scriminate between P magI/lis nnd P. lIIicros whereas standard manuals have re lied on cell size lO separate Ihem., ·6 However, PEP kits have their drawbacks:

Kits are often markeled as ' rapid ' because they are read fOllr hours after inoculat ion, but don't forget that it usually takes 48 hours to grow an adequate inoculum! 11

Reacti ons are often qualitative, making tes ts difficult 10

read and prone to va ri ations in inocul um. It is important lO standardise the inocu lum and to use colour charts to make endpoints easier to read. I

The databases often do not include all the recognised species. They are also unavoidably inaccurate as they have to incorporate geneticall y heterogeneous species slich as P prevolii for which identilkat ion criteria are ullclea r.

This problem arises because the classifi cation of the butyrate-producing spec ies such as P Clsacc/wlVlYlicus and P prevO/ii is very unsa tisfactory. I PEP kits are highly di scriminatory, and when they are used to characterise

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f igure 3. Comparison of Prclonncd Enzyme Proli les (PEPs) of four most impon'lIl t spcci!!s. P //IagllUs. I~ lIIicl"O,\', I~ (I.ml.'dU/rolylic" .~ and I~ (l/wemiJill.v.

strains which belong to undescribed species, the database wi ll inevitably be wrong. As the classification is refin ed and new species are described, the proportion of strains whi ch can be identified to adequately de fined spec ies will increase. I r a clinical isolate docs not fi t clearl y into a recogni sed spec ies, then it should be reported onl y as 'Pep/OSI replococClfs species" .

PEP kits must be interpreted in conjunction with the cell and co lonial morph ology, which are lI se fu l guides, parti cularl y for I~ al/aeroi; ill s, P. II/icros, and (w ith experi ence) I~ ascl(:charolyticlfs. Colonies are best Gram fi lmed after 48 hours' incubation, as younger colonies arc often 100 smal l and cells from older colonies tend to become ple01110qJhic and to decolourise rapidly. A good sense of smell is va luable for P. tlllaerobiw' and some slnl ins of P. asaccllmvlyticlIs, as well as rarer species slich as P. hydrogel/a lis. Testing for sensit ivity to sodium polyanelhol sulphonate (SPS) is highly sensitive and specific for P. al1aembills; SPS discs are cOlllmercially available and very use ful. A n ident ification scheme ror GPAC has reccmly been published. I

Clinica l importa nce

GPAC are part of the normal fl ora of the upper respiratory and gastro- intestinal tracts, female gen ito-urinary system and skin: 12 they account for about 25-30% of all anaerobic isolates in human infections ancl arc particularly associated with abscesses, so ft-ti ssue and obstetric infecti ons.1. 13 When they are isolated in 'heavy mi xed growth ', it is diffi cult to dec ide when they are acti ve ly pathogenic, i.e, contributing to the infecti oLis process, or are merely ' passengers'. However, several specie.'), nOlably P magmlJ, h,l ve been isolated in pure culture frol11 infections such as superfi cTa abscesses, prostheti c j oint infecti ons and , rarely, from l ife­threatening in fec tions including endocarditi s. I

The mouth is host to variable numbers of P. micros and I~ allaerobiils, P. micros is now recognised as an important pathogen in oral infections such as severe periodontitis'4 and peritonsillar abscess. 15 GPAC are the commonest anaerobes in superficial abscesses and skin-related in fecti ons, for instance non-puerperal breast abscess,16 diabeti c foot infecti ons, 17 and wound infecti ons.1.8.13 They can cause low-

3

Culture Vol 20 No 1

grade septic arthri ti s after orthopaedic procedures , parti cularty after replacement of the hip and knee.2,l s Their place in the pathogenesis of bacterial vaginosis is less clear but they have been much less studied than GNAR; I they are well recognised to calise obstetric complicati ons such as chori oamnioniti s and puerperal bacteraemia. ' Otherwise, GPAC are rarely iso lated from septi caemic episodes or in fecti ons of the CNS other than brain abscesses.

Pathogenesis

This subject hns been very poorly slUdied. As a general rule, infecti ons related to skin or mucosal surfaces appear to in volve GPAC known to colonise that surface, indicating that infecti on usuall y arises from the endogenOlls f1 ora. 12

Studies in the 1980s indicated that capsule format ion was an important viru letlce factor, 19 but the species identity of these strains was not reponed. Recently, 'rough' strains of P. lJIicros20 have been recognised; they appear to possess fibrillar structures which may be related to adherence. Several potential virulence factors of P. /JIagl/lls have also been described.21.22

Antib iotic sensitivity patterns

The great majori ty or cl inica l isolates are susceptible to metronidazole and penicillins; I. 23 ~- I actalllase production has never been adequately documented. Vancomycin, chloramphenicol and imipenem are also highly effective but susceptibi lity to clindamycin, macrolides and cephalosporins varies. l There appear to be considerable differences between species in susceptibility pa'tterns; 1.23 regrettably, most reports have not attempted to present data for indi vidual species.

lnd ividua l species

P II/agllils is the most important species of GPAC, in terms of frequency (il accounts for about a third of all cl inical isolates) and virulence; I there are several case reports of fata l infections in previously well indiv iduals. It is probably the commonest ,tIli.lerobe in superficial abscesses8,16 and soft-ti ssue infections, pan icularl y of diabetics;17 it causes low~g rade in fec ti ons of prosthetic joints2•18 wh ich are diffi cult to diagnose and probabl y under·recorded. Unlike other GPAC, il is niore often cultured with aerobes,S notably S/(Iphylococci/s Cl l/reus from skin ulcers and boils, than with other anaerobes. Unfortunately, growth on blood agar is often slow and many isolates wi ll be missed if plates are di scarded after 48 hours' incubation, particularl y as colonies are pleomorph ic and not distincti ve. Likewise, cell morphology is extremely variable, Standard manuals offer no satisfactory tests for separat ion from P. 11IiCIVS5,6 but PEPs afford a simple and reliable means of identification. 1 There has been some interes ting work on the range of potential virulence factors which P. maglll/s forms; prOduction of extracellular enzymes such as collagenase has been correlated with site of isolation,21 and the formation of Protein L, a factor ,>vhich binds !O immunoglobulin Jighl chains, has been linked to lower genito-urinary tract infections.22

P. miclvs is rarely reported from ex tra~o ral sites; i t is probab ly mi ssed because il is a fastidious anaerobe which usua ll y takes 5 days to appear on ordinary blood agar. Oral microbiologists have recently shown much interest in its role in periodontitis and dental abscesses,14 and it can often be isolated from mi xed anaerobic deep organ abscesses (e.g. of brain, li ver, lung) with organ isms such as 'Streptococcus mil/eri ' and fusobacteria.24 The cell morphology reveals small Gram-pos iti ve cocci, usually arranged in chains

Culture Vol 20 No I •••••••••••••••••••••••••••••••

Figure 4. Cell morphology of lyrical Slra/1I of R miclV.r ,Ilier 2 days' inclIv,llioo.

(Figure 4). Most strain s form whi te, raised coloni es surrou nded by a characteris ti c ye ll ow-brown hal o of discolouration in the agar, which give a highl y distinctive appearance. Testing for alka line phosphatase formation to distingui sh from P. lIIagJ/uss is unreliable, but P. micro.\' forms a wide range of proteolytic enzymes and is easil y ident i fied by its PEP,I.II A selective medium has recently been described.25

P. (fsacc/wro/yliclIs is the most important member of the large group of species which form the VFA butyri c acid as their terminal YFA. 1•11 The classificat ion and identification features of this group arc poorly defined and many reports in the literature arc likely to refer to misidentifications, but P. asacchamlyliclis is sometimes isolated in pure culture from important specimens such as abscesses.8 Its identification is contentious: most but not all authorities consider that all strai ns form indoie.1.5.6 With experience. the Gram fi lm (s trongly deco lourised cocci in clumps, looking like neisseriae) and colonia l morphology (convex grey-yellow colonies afler 5 days) arc very helpful (Figure 5).' About 25% of strains produce a characte ristic ' mouse's cage smell' li ke veillonellae. PEPs must be interpreted with care as a recently described species, ' /~ "arei', produces a very simi lar profi le; the two species are easil y separated by cell and colonial morphology.l.'!

In contrast , P. Cl IIClerob ills is easy to identify. I The Gram film lIsually reveal s coccobac illi in chains with marked pleo morphi sm. Isolates grow rapid ly to form 1-2mm colonies after overnight incubation; they usuall y have a disti nctive sickly-sweet sme ll like a child 's toy phlsticine. Identification using PEP is simple but rarely necessary; SPS discs are cheaper and as reliable. It is al so the onl y spec ies of GPAC to form the YFA isocaproic ac id . However, the pathogenicity of P. clIlaembius is less clear-cut than the

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Figure S. Cell morphology of lypic:l I strain of P asru:dlllro1.wic/is arIel' 2 days' incub;llion .

4

species discussed above: 1 it is part of the gastro-i ntestinal flora and is often isolated in heavil y mixed cultures from sites such as leg ulcers,8 but it may be more of a passenger than a pathogen; it has rarely been recorded in pure culture. It is much more resistant to antibiotics than other GPAC; some strains are resistant to metronidazole and others to a wide range of B- Iactams, which may include cefox itin and cefotaxime.1 Severa l lines of evidence, notably the lack of effect of B-Iactamase inhibi tors, indicate that the mechanism probably does not involve B-Iactamase production. I

Areas which would repay further study

GPAC have been very neg lected and several aspects merit research programmes, for instance the classification and identification of the butyrate-producers and the who le area of palhogenes is. However, there is plenty of scope for small projects to yield val uable information 0 11 their biology. A few ideas for interesting projects: • Aerotolerance - almost no in forma tion is avai lable

• Metabolism - There appear to be no studies 011 the growth requirements of indi vidual species. e.g. P. micros.

This could lead on to:

• Development of selec ti ve med ia. GPAC arc susceptible to IllOSt antimicrobials, but possible selective agents include bicozamycin , novobiocin, aztreonam and oxolinic acid. I

• Transport media - few recent data 0 11 effectiveness

• Identification of GPAC found in

leg ulcers the norma l fl ora (part icul arly the ski n)' the fe male genito-urinary tract

(though it is likely that many strains from these sites cannot be identified (Q recognised species)

• An ti biotic res istance -how common is metronidazole resistance'll mechani sm of resistance of P. allaerobilfs to ~- Iactams

Whenever possible, reports should idel1li fy isolates to the species level- too many previolls reports have grouped them all as ' PeploslreplOcocclIS spp.', greatly reducing their usefu lness.

References I. 1vlunloch. D.A. (1998). Clil!. Mkmbi(ll. Hel' .. II: 81 - 120. 2. Bourgault. A. el rd. (1980). 1\1111. 1111. Ml'd .• 93: 244- 248. 3. Collins. t-.'I.D. el al. ( 1994). 1111. J. SYSI. IJllcleriol.. 44: 8 12- 826. 4. Murdoch. D.A. 1'1 al. ( 1997). 1111. J. Sy,I·I. IJ(I£'leriol .. 47: 78 1- 787. 5. Summ:men. P. el (II. (1 993). W:l{lsworth Anaerobic Bacteriology Manual. 5th cd n.

Star Publ i ~hing Co .. LO$ Angcles. USA, pp. 78-81 6. Hi llier. S.L. and Monel<l. BJ. ( 1995). In: rvl:lnual ofClinic:,1 Microbiology. 6th

edn. Murray. P. R . ('1111. (Eds). ASM Press. W:lshington, USA. pp. 587- 602. 7. WaU. B. and J<lck. E.P. ( 1977). J. Ml'd. MicmiJiv/ .. 10: 461-468. 8. Murdoch. D.A. ('1111. ( 1994). J. Med. MicmlJiol .. 4 1: 36·44. 9. Wren. M.W.D. ( 1991 ). CIIIIIII~'. 12: 2-4 .

10. Hegi nbolhom. M. ('I (II. ( 1990). J. Clill. />mhol.. 43: 253- 256. J J. Murdtx:h. D.A. and MiJchclmore. U . ( 1991). J, MI'd MicmhioL 34: 295- 308, 12. Finegold. S. M. and George. WL. ( 1989). Anaerobic infections in humans.

Academic Press. NcwYurk. USA . 13. Brook. l. (1988). J. C/;/I. MicmiJi()/ .. 26: 118 1- 11 88 . 14. Ituns. T.E. cl a/. ( 19<)2). Orol MiaoiJiol. 111/11111110 1 .• 7: 1- 6. 15. Mitehclmorc. I.J . 1'1 III. (1995). fill: J. Clill. Micl'Ohiol. II1/ecl. Dis .. 14: 870·877. 16. Edmiston. c.E. 1'1 al. (1990). J. Ill/ect. Dix .. 162: 695- 699. 17. Wheal. LJ . 1'1 (1/. (1986). /\,.('/1. 1111. MI'd •• 146: 193- 1940. 18. Fitzgcrald. lUI. 1'1 (I/. ( 1982). C/ill. OnllOpet/.. 164: 141 - 148. 19. lIrook. I. and Walker. R.1. ( 19R5). Call. J. Micmhiol .. 3 1: 176- 180. 20. van O:llcn. PJ. ('/ III. (1 993). 1111. J. Sy.l'l. BaCluiol .. 43: 787- 793. 21. KrellCl. c.J. I!IIII. ( 1991). J. 111/l'cl. Dis .. 163: 1148- 11 50 . 22. Kastc::rn. W. /'1 (II. ( 1990). III/ecl. IlIIl/lIIll .. 53: 121 7- 1222. 23. Bowker. K.E. ('I til. ( 1996). I Alllilllil:lViJ. Chell!()/hl'I:. 3M: 27 1- 28 1. 24. l\'lu rdoch. I).A. /'1111. ( 1988). IJ lllcel. i: 594. 25. Turng. B.F. el al. ( 1996). Oml Micmhiol. Il/lllm'lol .. II : 356- 36 1.

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Culture Vol 20 No I

Vibrios on the half- shell

J. Glenn Morris, Jr., MD, MPH 8:TM Professor of Medicine

Un iversity of Maryland School of Medicine, Baltimore, Maryland, USA

Strains of the genlls Vibrio are ubiquitolls in the es tuarine environment where they are free-li v ing (autochthonous) organisms. Their presence does not correlate with faecal contaminati on I; if any thing, the re are suggest ions that vibrios arc morc cOlllmon in clean, "pri stine" areas away from sewage outflows. As might be expected, gi ven their e nvi ronmental d istribution, vibrios arc also a freque nt isolate frol11 seafood (und. in particular, she ll fi sh). For example. in our Chesapeake Bay studics2, we found that Vibrio v lllllijiC/lS alone constituted approx imately 8% of the lo tal cuhurablc heterot rophic bacteria present in Bay water during mon ths whe n wate r temperatures were >8°C; when V. va/lli/icus was present in water, it was also present in oysters, w ith coun ts in oys te rs exceed ing those in surroundi ng harvest wa ter by two orders of magnitude.

To date, strains from 12 vib rio species have been isolated fro l11 humans (Table J? The clinical significance of some of these isolations is uncerta in. Vibrio carc/wriae, for exnmplc, h:ls been iso/aled only once, from a wound resulting from a shark bite". In the absence of a drmnatic increase in thc numbe r o f humans being bi llcn by sharks, V. carc/wriae should nOI warran t maj or attention from cli nic ians. In contrast, Vibrio cho/erae. Vibrio parali aemo/Yficlls, and V. vU/ll/jicus a re un iversnll y recognised as important human pathogens; wi th Vibrio hollisae, Vibrio a/gillo/yficlIs, Vibrio p uvialis, and Vibrio mill/fCIIS each responsible for a sma ll er, bu t substantial , number of cases. Data on frequency of iso lat ion of these pathogens in the state of Flo rida fo r the years 198 1- 1993' are summarised in T"lblc 2. In this laller study, 87% of pat ients for whom a food his tory was ;.lVail<lble reporlee! eating seafood in the week before onse t of illness; 87% of those ea ting seafood had ca te n raw sea food, and 82% had eaten raw oysters. While this study did not include a contro l g roup, there have been several smalle r U.S. studies in which vibrio infec tions have been significantly assoc iated with raw oys ter consulllption6-8.

While these linkages between the es tuarine envirollment, seafood, and human discase are cOlll mon among vibrios, each species has certai n dis tinc ti ve reHllIrcs. Data and issues are sUlllmarised be low for the three major pathogcnic species.

V cllO/erae

V. cI/O/erae is the aet io logica l agen t of cholera, a de vastating dise.lse that C<.l n cause severe, dehydnll ing diarrhoea and dea th in healthy adult sY. However, the species V. choferae is hi ghl y di verse, with human illness associated with a single, almost clonal group of strains characteri sed by the presence of CTX$, a s ingle-s tranded DNA fi lamentous bacteriophage which encodes cholera tox in (CT) IO, and a Cil 40 kb vibrio pathogenicity is land (designated VPI) which encodes TCP (the receptor for CTX$) and associated colonisati on factors (the TCP/AC F gene c luster)ll . These "epidemic" strai ns

5

have tended to be withi n V. choferae 0 group I (o r, more recent ly, 0 group 139). However, there are 0 I strains which lac k these critical genctic ele ments , and which do not appear to be pathogenic.

CT-producing, "epidcmic" 0 I V. cho/erae strains can be isolated from the e nvironment. In Peru 12, we have shown that coun ts of CT-produc ing strains in the environme nt correlate s ignificantly with the number of cholera cases reponed in the community two months later; the initia l rise in cOllnts in the environment appears 10 be linked with increases in wa ter te mperature_ As cases begin 10 occur in human popu lations, there is then the po tential (particularly in developi ng count ries) for secondary amplification, with microorganisms from infected persons further contaminating the envi ronment and food and water sources, leading to epidemic disease. T he organ ism may be transmilled fro m the environment to humans by a number o f rou tes, inc luding consumpt ion of undercooked seafood or she ll fis h: in the South American epide mics in 1991 , ceviche (a marinated, raw fi sh di sh) was initia lly implicated epide miologicall y as a vehic le of transmi ss ion, while crabs have been repeatedly implicated as the SOurce of U.S. Gulf Coast-associated cholera cases J 3.

V. cho/erae stra ins which lack the genetic po te ntial fo r causing e pidemic d isease have generally been referred to as 11 0 11 -0 g ro up I , Or n on ~O J , V. c/lO feraeI4 . No n-O l V. choferae are ve ry common in the envi ronme nt, and show a great dea l of in traspeci fi c variabili ty, comparable to that seel1 among members of the species E. coli. Certain s tra in subgroups are potentia ll y pa thogen ic: slrai ns which produce a specific hea t s(able enterotox in (NAG-ST) have been linked with occurrence of gas lroente riti s I5•16, while strai ns which have been isolated from patients with bacteraemia are generall y heavily e ncapsulated. It is like ly that most o ther

T:.tb lc I. Vibrio species impl icatcd as II C:1l1se of human di seasc

V. cho/erae

V. jJarallClemofYl iclIs

V. jlll via /is

V. mimiclis

V. holti.me

V. ji /missii

V. VU/l1 ijiCIiS

V. a/gillo/yticlIs

V. dOll/se/a

V. cillcil1l1atiellsis

V. carc/wriae

V. lII el .w:/lIIikol'ii

J

Ii I

I:

I

Culture Vol 20 No I •••••••••••••••••••••••••••••••

non-O 1 strains pose a mi nimal risk to humansl4 . In work done by the U.S. Food and Drug Administration, V. cholerae non-Ol were isolated from 14% of freshly harves ted oyster IOLSI7; however, in the absence of a clear method for din:erelllinting pathogenic from non-pathogeni c strains, it becomes very difficult to assess the cl ini cal significance of these isolates.

V para/lOemo/yticLiS

V. paraliaemo/yliclis is the most commonl y isolated " noll­cholera" vibri o. As shown in l )lble 2, it constiwted 30% of all vibrio isolates in Florida between 198 1 and 19935. In Japan , it has been implicated as the cause of at least a quarter of tota l foodborne disease cases. In <1 Ca lcutta study, rates of isolation of V. paraliaemo!yliclIs varied between 3.5 and 23.9% of patients acutely hospitali sed w ith diarrhoea; in a study in Bangkok, an iso lat ion rate of 19% was reported among adults with diarrhocal illness presenting for care at a local hospital IS. For reasons which are not completely clear, there has n OI been the strong epidemiological association betwee n V. parahaelll o /y liclIs cases and raw oyster consumption which has been descri bed for 110n-0 I V. c!/O!erae and V. vlI/llijicIIS. However, in outbreak studies, infection has consistentl y been linked w ith consumption of seafood.

The pathogenicity of V. paraliael1lo/ylicus isolates has traditi onall y been correlated with the production of the thermostab le direct hacmolysin (Vp-TDH ) which is responsible for the beta-hacmo lys is seen when these isolates are plmed on Wagatsum<1 agar P). Origina l studies in Japan showed that this phenomenon, named the Kanagawa phenomenon after the prefecture in Japan where it was di scovered, was present in 96% of clinical isolates but on ly I % of environmental isolates. Volunteer studies also demonstrated the importance of Vp-TDH: Kanagawa­positi ve strains prod uced diarrhoea whereas doses of up to 109 Kanagawa-negati ve stra ins failed to do so in 15

vo lullteers. Furthermore, studies w ith isogenic mutants have shown that deletion of the Vp-TDH gene results in loss of emerolox ic acti v ity in laboratory models (Usi ng chamber and rabbit ileal loop assays)20.

In recent years it has become clear that the pathogenesis of V. parahae11loiyriclfs is more complicated than orig inal ly thought. A second group of haemolysins, known as Vp­TDH-related haemolysins or Vp-TRH, can be found in cerlain clinica l isolates, especiall y those that are Kanagawa­negati ve. These haemolysins are genetically related to Vp-

Figure 1. Growth of V. dlOfcmc (on right) and \~ I'lIfllijiCI/S (on left) 011 th iosulph:uc citratc bi lc-salts sucrosc (TellS) selective mcdium: notc the characteristic ycllow colour of thc V. cho!emc colonies. and thc blue-green colour of \~ )llIfllijims on this med iulll.

'1\Ibic 2: Cl inica l syndromes assoc iated with Florida vibrio infections, 198 1- 1993 ( from Hl ady and Klontz5)

Number of isolates, bl' cl inical »I'cscntation Species gaslt'o- primary wound other unknown TOTAL

enterit is septicaemia

V pamhaelJlo/yricw; 120 (58%) 16 (8%) 56 (27%) I ;! 13 (6%) 206

V. vull/ijicus 16 ( 11 %) 75 (53%) 47 (33%) - 3 (2%) 141

-V choleme (non-O l ) 87 (67%) 20 ( 15%) 11 (8%) 4h (3%) 8 (6%) 130

V. I /O/lisae 50 (85%) 3 (5%) 4 (7%) - 2 (3%) 59

V. a/gillolyliclIs 6 ( 12%) - 37 (7 1%) 6'(12%) 3 (6%) 52

V. fl il vialis 35 (73%) 3 (6%) 5 ( 10%) 2d (4%) 3 (6%) 48

V. lIIill/iellS 34 (85%) 1 (3%) 1 (3%) I" 3 (8%) 40

V dalllse/a - 6 ( 100%) - 6 ... V. lIIelcllllikovi i 1 ( 100%) 1

TOTAL 348 (5 1%) 11 8 (17%) 168 (24%) 14 (2%) 35 (5%) 683

" urinnry IraCI infecti on b 2 pu lmonary infections of drowning vict ims. I car infection. I urinary IraCI infectioll "2 pulmonary infections of drowning victims. 2 car infecti ons. 2 urinary Imel infect ions d gall bladder infec tion

6

TDH (they share around 70% seque nce homology) but are more diverse «2.8% divergence among mos t Vp-TDH versus 16% between 2 subgroups of Vp-TRH). Urease production has recently been identified as ano ther possible virule nce factor (or marker fo r virulence), and may be especially important in isolates from the Pac ific coast of North Amcri ca21.22.

V vulnificus

\~ VIIIl/ij/CIiS is a recentl y identified vibrio species which can ca Li se woun d in fec ti o ns a nd " prima ry septicaemi a" (septicaemia resulting from inges tio n o r the organism, without any o ther obvious source, such as :J wound?·R.23.24. It is the leading cause of she llfi slH.1ssoc iated deaths in the United States. Whi le incidence da ta are limited, it also appears to be a substanti ve c linical problem in Korea and Taiwan. V. VII/llijiCIIS septicaemia occurs almost exclusive ly among persons who have underlying liver d isease, are alcoho lic, o r a re imlllunocomprom ised . One third or pati e nts w ith V. \llIllI~ficlI.\· septicaem ia preseill in shock. Thrombocytopenia is common. and there is o ften evidence of dissem inated intnIvascula r coagulation ; gastroirllestinal bleeding is not infrequent The mortality r;Hc is >50%, with morta lity exceed ing 90% among patienls who become hypotensive within the first 24 hours of hospita lisa tion. The re is a strong epide miological link between ,~ Vll/l1~ficflS septicaemia a nd eating raw oysters7.8.

Virulence of V. vU/llijicllS has been associ ated with production of a polysaccharide capsule, and there are suggestions that certain capsule types are more likely than others to cause human di sease25 . However, at thi s time there is no way to clearly differentiate po te nt ially pathogenic rrom non-pathogen ic strains - nor is there any certainty that there nrc, indeed, non-pathogenic V. vulilijic// s. Thi s c reates major problems frolll a regulato ry standpoint , as virtually all sUllllllcr-harves ted oyste rs from the U.S. Gulf Coas t and the Chesapeake Bay appear to carry the orgnnism. Using data from the Florida Behavi oral Risk Factor Survey and health department surveillance data , the annual rate of V. vlI/lI~ficu.\'

infec tion among persons with li ver disease (i.e, the major populat ion group susceptib le to in fec tion) who ate raw oyslers was es timated H( 72 cases per J mi ll ion adu/(s26. This rate is much lower than migh t be expected ir all V. vU/llijicu.\" prescnt in oys ters had an equal ri sk of caus ing illness, sugges tin g thai th e vas t major it y o r oys te rs carry ing V. vulllijicl/s do not rcpresent a health risk. However. given the severity or V. VI/III~ficlis infec lion, and in the absence of

Culture Vol 20 No 1

•

FiJ.:ll r<! 2. Restllts of screening of a colony blot of an environmcntal sample with an alk aline phosphatase. labcl1cd oligollucleotide probe for V. vll lll ijiCIIS. The satllpk W;lS plated on a llon·selcctive medium and incubated overnight : hybridisation. as indicated by a dilrk colony on the blot. is indicative of the presence of \~ I'IfI"ijino.

any method to differentiate "safe" from " unsafe" oysters, it wou ld appear prudent for persons with underl ying liver disease and o ther ri sk f~lc lors ro r in fection to avo id raw oyster consumption.

Identification of the organism

Vibrio species are readily cultured on a variety of media, w ilh 1110st c urrent commercial identificat ion sys te ms ab le to differentiate the va rious ~pecies . Isolation of vibrios from stoo l or e nvironmental sOllrces is facili tated by the li se of selective media, suc h as thiosulphate citrate bi le-sa Ils sucrose (TeBS) (Figure I).

O ur laborato ry has pioneered the use of DNA probes for ident ifica tion and e numeration of vibr ios from environme ntal and rood sources2. 12.27.2S. Briefly, water samples o r oyster homogenates are pla ted di rectly on non­selec tive Inedia (such as Luria agar), incubated overnight , a nd co lo ni es transfer red to an appropriate fi lter for hybriclisa tion. We gene rally use alka line phosphatase­labelled ol igonucleotide probes, which permit defi niti ve identificat ion of colonies of interest wi lhin a matter of hours. We have co.nsistent ly shown thi s methodology to be much more sensitive than conventional methods. Sensitiv ity is furth er e nhanced by Ou r li se of non-select ive media: vibrio coun ts obtained from L-agar are almost always two orders o r magnitude highe r than those obtained when

Chromogenic un Medium (CM949)

Oxoid Ltd has introduced new Chromogenic UTI Medium (CM949) to i ls range. This medium is for Ihe presumpti ve identificat ion and differentiation of all the m;:\in micro-organisms that cause urinary tract infect ions (UTI 's).

It conwins IWO specific chromogenic substrates which are cleaved by enzymcs produccd by Ellrerococcus spp .. E. coli and colifonns. It contains pheny la1{!ninc and tryptophan which provide an indication of tryptophan deamin<lse activ ity, indicating the presence of Proteus spp .. Morsal/ella spp. and Providel/cia spp. This formulation allows positi ve idcntific<.ltion of individual target organisms, by provid ing clear di fferent iation between colonies in mixed culturc.

\ \.

t ...

'~. ~~.10

By providing specific presumpti ve idcntification of target micro·organisms. Chromogenic UTI Medium saves laboratory time by reducing the need for subculturing or further diagnostic tcsting.

Forfu,.r"er il/formorioll COl/flICI: Valerie Knne. Oxoid L imi ted. Wade Road. Basingstoke. Hants RG24 SPW. England. Tel: (0 1256) 841 144. Fax : (0 1256) 463388. c-mail : Oxoid @oxdgb.sprint.com

7

Culture Vol 20 No 1

identica l samples arc inili c:llly plated on Te BS. Use of this methodology permits d irect identi fica tion of strains carry ing critical vi rulence factors, sllch as CT (for V. cI/O/erae) or Vp­TDH ( for V. paralwemolyliclIs) . Our V. Vl/l"ijiCWi probe is now routi nely lIsed by the U.S. Food and Drug Adm inistrat ion in environmental screeni ng. and thi s and other probes are findi ng increasing appli ca tion in environmenta l research (F jgure 2).

Summary

Vibrios arc ubiq uitous in the envi roll1l1CIH, and a common "contami nant" of shell fi sh and other seafood. The clin ical signin c<lnce of vibri os isolated from seafood is variable. Strains which are CT- or Vp-TDH-posit ive represent a c lear public health r isk. Other isolates may or Jllay not represent a ri sk to humans, although the isolation o r a pathogen such as V. VUlll ijiCII S shou ld generate concern, given the sever ity or the ill ness it is capable or ca llsing in susceptible persons.

References

I. K"IX!r J. Lockmau I I. Colwell RR. jo.'~cph SW. Ecology. ,cmlogy. and cntcrotoxin production of I';/lr;o dlolel'll!' in Ches.1pc"kc Bay. IIf1J1I Em'iron Microbiol 1979: J7: 9 1- 103.

2. Wrighl I\c. Hill RT. J()hn~on JA. Roghnl.1n 1\-I-C. Colwell Rl{, I\-Iorri, JG Jr. Di'lribution of Vibrio '-II/lIiji'·II.I' in Ihe Chcs'llX!ake Bay. t\/iJI/ I~·n";mll Mirmbiof 19%: 62 : 717- 724.

3. Janda JM. Powers C. Bryant RO, Abbon SL. Current lX!r~ l)cclivc~ 011 Ihe epideilliology and pathogenesis of r.:Iinic:llly signific:ul\ vibrio specie .... Oill Micmbio/ HI'" 1988: I : 245- 67.

4. Pavia AT. Bry,m JII. Maher KI~. I - I e~ter TR. F:mner JJ Ill . I'llifio ClI/l'hwirw infeclion :Iner a :-hark bite. AIII/ 1,,1('1'11 Med 1989: I I I: 85- 86.

5. HI;Idy WG. Klontz KC. The cpidemiology of vibrio infec l ion~ in Florida. 11)81 -1993. J llIii-'t' l JJil' 1996: 173: 1176- 83.

6. Morri~ JG J r. Wihon R. Davb IlIt Wachsmuth IK. Riddle CF, W,lIhen HG. I'ollard RI\. Blake I'll . Non-Ol Vibrio dllJfcme gas1rocnteriti .. in thc Uni ted Slate~: CliniC:11. cpidemiologi(·. and laboralory cil:lr.,cteristics of ~porml ic ca~c~.

,t 'lll 11111'1"11 Mer! 1981: 9~ : 656-658. 7. John~ton JM. Beckcr SF. McF,wl;lIId LM. 'Vi/"io '·/lll/iji(·II.~: 1\bn :lIId the ~ea.

JAM,t 1985: 253: 2850- 2853. 8. T.,ckel CO. Brenncr F. BI:tke I'A. Clinical fe;lIures :md:H1 cpidemiologic :-Iudy of

Villl'ill '·/Ill/ijit·/1.~ infecliorl.~. J II/Jul Di.I' 198.J: 1 ~9: 558- 561. 9. K:lper m. Morri~ 1G J r. Lcvinc MM . Cholera. Clill Miemhilll ReI' 1995: X: 48- 86.

10. W:t ldol' MK . Mek;ll:mo~ JJ. Ly~ogenic conver~ion by a fil:1II1elllou~ ph:lge

encoding cholerJ lOX in. Sd!'I/CI' 1996: 272: 1910-14. II . K:tr:toli:- DKR. John~on JA . Bailey CC. llocdekcr EC. K:,pcr Jil . Recves PRo A

l'ibl'ill cllO/I'rtl(' p'lIhogcnicily isl:llld a:-~ociated with epidcmic and pandemic :-Ira in~. Pm NtIIl Actlll Sci USA 1998: 17: 3 134-3 139.

12. Fr.III(.'O I\A. Fix AD. Pr.:Kl:1 A. I'an .. dcs E. I~tloillino JC. Wrig.ht AC. JOhll;,()ll JA. I\kCru1er R. Guem, H. Morris JG Jr. O)()lem in Lima. Pcnl. correlmcs with prior isolmion of Vibrio choh'ml' from the environment. ,t", J q,i(lelllio! 1997: 140: 1067-75.

13. Lin FYC. Morris JG Jr. Kapcr m. Gro~~ T. Michal.~ki J. Morrison C. Libonali JP. bmel E. Persi~lence of cho1cr:t in the Uni ted St:ttcs: bol;ltion of lfi/}r iu c/w/era(' 0 1 from., patient wilh diarrhea in Maryland . J Clill Mit:roiJiu/1986: 23: 624-626.

14 . Morris JG Jr. Non-O group I Vi/)";o ('Iw/erllt': a look ;It the epidemiology of HII

occ:t~iollal p"thogen , EjlitJclllio/ RI'" 1990: 12: 179- 19 1. 15. Morrb JG Jr. Takeda T. Tall BD. Lo~ollsky GI\, I3tmtt:lc1mry:, SK. Forrest BD.

Kay !lA. Nishibuchi M. E.~pcril1lental 11011-0 group I I'i/Jrio c/w/cr(lC gastro­cnteri tis in humans. J Clill Im·f'.11 1990: X5: 697- 705.

16. Bagchi K. Echevcl'ria P. A"hllr JD. Selhabutr 0 , Serich:ullll1cl'gs O. I'[oge CEo Epidemic di:1I'fhOCll cauM!d by I'i/Jrio t:/w/c/'(/(' non-Ol that produccd heOlt-slllble toxin :UllOng Khmers in:1 c:unp ill 'l1milanu. J e lill Mh:mbiol 1993; 3 1: 13 15- 1317.

17. Twcdt RM. Madden JM, Hunt JM, Fr:Hlcb DW.Peeler rr. [)Ur:1II AP. Hebert WOo McKay SG. Roderick CN. Spite GT. Wazen~ki TJ . Ch"rac l eri~.tI1 i on of Vii)";/) ("/101('/'(1(' i ~o laled from oy~ters . ' \ 11111 Em'iroll MicmlJio11981: 41: 1475- 8.

18. Echc\'crria P. Pitarangsi C. E:Ullpokalllp 11. Vibulbandhilkil S. Boomhlli I~ Rowc O. A longiludinal sludy of the prevalencc of b<lc tcri;tl cllleropathogelis among adults wilh di:lrrhe:t in Bangkok. ·111ailand. Djo};" Microbiol llif('ci Dis 1983: I: 193- 204.

19. Jo~cph SW. Colwell RR. Kapcr 111. llil,';o f/(//'(//WCIIIO/yficlfS ,nld rdalcd halophilic vibrios. eRe Cril Rt'I' Miao/liol 1983; 10: 77- 124.

20. Nishibuchi M. F;tsano A. R\I~~II RG. K<lpcr m. ElllcrotoxigcnicilY of Vibrio l}(Imlw('lIIolylinf.f with ;lIId without gcncs encoding thcrmO!>\:lblc direct hCllloly~in. ilifi.'(·f /111/111111 1992: 60: 3539-45.

21. Kelly MT. Stroh EM . Urc:t:-c-po~ i tivc. Kanagilwa-ncg:lIi"c Vibrio /1I/mh(f('lIIolYlit'//s from p:,ticnt~ :md Ihc cll \'ironmcnt in lhc Paci fic Northwest. J Oill Mi('fobio/1989: 27: 2820- 2.

22. I\bbou SL. Powers C. Kaysner CA. Takeda Y. l ~ hib;t~hi ""1. Joseph SW. landa JM. Emcrgence of a restrictcd bioscrovar of Vibrio jJtI/"(I/r(lClllolylicIfJ as thc prcdomin;ml causc of I'ibrio-:t~,~ociatcd g:tSlrocntcritis on the We~ 1 Coast of the Ull ited Swtes :md Mexico. J elill Micmbiol1989; 27: 2891 - 3.

23. KIOII\I. KC. Lieb S. Schreiber M. J:lllow~ki HT. !laldy LM. Gunn RI\. Syndromes of Vibrio 1·llflli/it'II.I· infec tions: Clinical and epidemiOlogical fe.Hurcs in Florida c:tscs. 1981-1987. 1111// 11111'/'11 Mf'(/1988: 109: 3 18- 323.

24. Park SD. Shon I-IS. Joh NJ. Vibrio m/llijiclIs septiccmia in Korea: Clinical and epidemiologic findings in scvcnty p:ltient~. J A/II 11 ('(/// Dcrlll 1991 : 24: 397-403.

25. Hayat U. Reddy GI'. Bu~h CA. John~on JA. Wright AC. Morris 10 Jr. Capsular t)'pe ~ of lfihdo "UlllijiCIU': I\n allaly~ i ~ of strains from clinical and cnvironmcntal ~ource~. J /Jifi.'Cll)i,~ 1993: 16H : 758- 62.

26. Ccntcrs for Disease Control. I'i/Jrio ,.,tlnijic//s inf('Clions :t~sociatcd wilh raw O)':-Ier con~umption - Florid:l. 1981-1992. M oriJirl MOr/tlI W('ckl,l' Re" 1993: 42 : 405-107.

27. Wright Ae. Miceli Gil. L:lIldry WL. Chri~ly m. Walkins WD, Morris JG Jr. Rapid identific:tlion of lfibrio \'If/llijit'Ul' on non-selecti"c media \\ilh ;111 alkaline pho~l'lmlasc-labcled oligonucleolide probe . AI'!'/ /;'1II'irml Micro/)j(J1 1993: 59: 54 1- 546.

28. Wright Ae. GIIO Y. Johnson JA. Nataro JP. 1\lorris JG Jr. Dcvelopmcnt .1l1d lc~t ing of ;1 non-radioactivc DNA oligonuclcotide probe that is spccific for I1brio ("/1/1/1'1'11(' cholera toxin. J Clill Miao/)io/ 1992: 30: 2302- 2306.

Till

Dryspot E. coli 0157 Latex test (DR 1 ~OM)

Oxoid LId has launched a novel. rapid and convenient latex agglut inntion [est for the ident ification of £. coli 0 157. The Oxo id Dryspol E. coli 0 157 Latex teSt (DR 120M ) can detec t 01 57 strai ns from culture wi th exceJ1 cnt sensit i vity and specifi city. proven in an ex tensive independent evaluation. •

E. coli 01 57 is the enterohaemorrhagic E. coli serogrollp considered Illost significant in human disease. causing a range of iJ1nesses from se l f-limiting watery diarrhoea to haclllorrhagic col it is (He) and haemolytic uraemic syndrome (HUS).

Each kit contains 120 lestS w ith latex reagents presentcd prc-dispensed on react ion cards. With a ll components suppl ied in dried format, the Oxoid Dryspot E. col i 0 157 Latex Test is uniquc in bcing able to be stored at room temper:uurc for lip to 2 years.

For /lfrther ill/orlllatioll COlltact: Valerie Kane, Oxoid L imited. Wadc Road. Basingstoke. Hants RG24 8PW, England. Tel: (0 1256) 84 1144. Fax: (01 256) 463388. c·mail: Oxoid@oxtlgb.sprint.com

Culture provides an international publishing forum for papers on microbiology. Authoritative artic les on microbio logical topics are welcomed. All submissions are peer reviewed by the Editorial Board. A synopsis of the proposed paper should

be sent to the Managing Editor of Culture at Euromed Communications Ltd ., The Old Surgery, Liphook Road, Haslemere, Surrey GU271 NL, England. Tel : 01428 656665. Fax : 01428 656643. e·mail: euromed@pavilion.co.uk

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