APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Dec. 1994, p. 4219-42250099-22401941$04.00+0Copyright X 1994, American Society for Microbiology

MINIREVIEW

Use of the CAMP Test for Identification ofListeria monocytogenest

R. C. McKELLARCentre for Food and Animal Research, Research Branch, Agriculture and

Agri-Food Canada, Otawa, Ontario, Canada K1A 0C6

SCOPE OF REVIEW

During the last few years there has been a veritable explo-sion of publications on the characterization of pathogenicfactors produced by Listeria monocytogenes and the animalhost cell immune response to L. monocytogenes infection.While this review will touch on some characteristics of viru-lence factors, its main objective is to summarize the literatureon the use and validity of the CAMP test for identification ofL. monocytogenes. Readers interested in a more detaileddiscussion of virulence factors and immune responses arereferred to the review by Portnoy et al. (55) and several recentpublications (16, 40, 76).

INTRODUCTION

Listeria monocytogenes, a ubiquitous food-borne pathogen, isof considerable concern to the food industry. It has beenresponsible for several outbreaks of listeriosis resulting fromthe consumption of contaminated food and continues to beimplicated in isolated cases of illness. Economic losses attrib-utable to listeriosis have been estimated at $480 million in theUnited States (56). Attempts to isolate and identify L. mono-

cytogenes and to limit its proliferation in foods have been thefocus of a significant international effort (19, 24, 32, 55, 57).One of the most important areas of study is the development

of rapid, specific methods for the isolation and identification ofL. monocytogenes. This pathogen shares distinguishing charac-teristics with closely related strains which are nonpathogenicfor humans, and thus it is often difficult to clearly establish theidentity of presumptive isolates (61). L. monocytogenes pro-duces a hemolysin which lyses erythrocytes (RBC) (55). Thisphenomenon has been utilized with some success as an iden-tification factor; however, many strains ofL. monocytogenes failto produce sufficient visible lysis on blood agar plates.An improved method based on the stimulation of lysis of

RBC by L. monocytogenes in the presence of other microor-ganisms producing extracellular enzymes has been identified.The resulting zones of lysis are larger and appear sooner, thusfacilitating the rapid identification of L. monocytogenes. Thistest (generally referred to as the CAMP test) has been adoptedas part of the official methods for isolation and identification ofL. monocytogenes by several organizations (23, 26, 75), in spiteof the fact that little attempt has been made to identifyextracellular factors of L. monocytogenes which may be in-volved in the reaction. This is particularly important in view ofthe controversy in the literature concerning the correct inter-

t Contribution no. 2235 from the Centre for Food and AnimalResearch.

pretation of the CAMP test. It is therefore appropriate tosummarize the varied reports on the synergistic hemolysisphenomenon and to assess the validity of the CAMP test as itapplies to virulence of L. monocytogenes.

HISTORICAL DEVELOPMENT OF THE CAMP TESTThe term CAMP test has been used to describe the syner-

gistic lysis of RBC in the presence of diffusible exosubstancesproduced by microorganisms growing adjacent to each otheron the surface of blood agar. It may also refer to similar testsusing partially purified exosubstances contained in wells in theagar or added to RBC in test tubes or microtiter plates. Themethod specifies that the microorganisms to be tested are to bestreaked in straight lines at right angles (90°) to one another onthe surface of blood agar, close to but not touching each other.After appropriate incubation, some lysis may be observedimmediately surrounding the streaks; enhanced lysis will ap-pear in the areas where the diffusible exosubstances of themicroorganisms overlap.The original CAMP test was described by Christie et al. (13),

and the test is named after those authors. They observed thatwhen hemolytic streptococci were inoculated onto the surfaceof sheep blood agar, enhanced lysis appeared adjacent tocolonies of beta-toxin-producing Staphylococcus aureus. Thissynergistic phenomenon, which was observed only with sheepand ox blood, was attributed to an extracellular, filtrable agentwhich showed considerable thermostability (100°C for 5 min).These workers further noted that only those streptococcibelonging to Lancefield group B gave a positive CAMPreaction, and they suggested that this test could be used as a

simple confirmatory test for this group.The term CAMP test strictly applies to the synergistic

reaction between S. aureus and group B streptococci as definedby Christie et al. (13). As this method has evolved to includemany other pathogens and test strains, the use of the phrasehas persisted. With a view to consistency, the term CAMP testor CAMP reaction will be used here to refer to any synergisticlysis of RBC by L. monocytogenes and any test microorganism.

DEVELOPMENT OF THE CAMP TEST FORL. MONOCYTOGENES AND RELATIONSHIP

TO VIRULENCEIn 1962, Fraser (27) was the first to observe the synergistic

lysis of sheep RBC by L. monocytogenes and either S. aureus orRhodococcus equi. In a more detailed study, Fraser (28)examined the abilities of a wide variety of pathogens tosynergistically lyse RBC. He concluded that L. monocytogenesgave enhanced hemolysis on sheep RBC or rabbit RBC with S.aureus or R. equi but gave enhanced hemolysis only on rabbit

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RBC with Corynebacterium ovis or Corynebacterium renale. Onthe basis of these studies, Fraser concluded that ruminantblood gave the most distinct and consistent results.

Further development of the CAMP test for L. monocyto-genes has focused on the use of S. aureus as the most consistenttest strain. Groves and Welshimer (31) were the first to observethat a positive CAMP reaction between L. monocytogenes andS. aureus (SA+ reaction) was related to virulence of L.monocytogenes. These workers tested 112 animal, human, andsoil isolates of L. monocytogenes and were able in all but onecase to link CAMP-positive, rhamnose-positive, and xylose-negative phenotypes to pathogenicity. These characteristicshave been used subsequently to identify virulent strains of L.monocytogenes and have been incorporated into several rec-ommended standard methods (23, 61, 75). It should be notedthat these methods require that L. monocytogenes give anegative reaction with R. equi (RE- reaction). In support ofthis, Schonberg (58) reported that L. monocytogenes did notreact synergistically with R. equi. He described several tests forvirulence of L. monocytogenes. In addition to experimentalinfection of mice and chicken embryos, he suggested that an invitro test for hemolysis, potentiated by S. aureus, is a goodindicator of pathogenicity. Since that report appeared, severalother laboratories have provided findings in support of theSA+/RE- reaction for L. monocytogenes (4, 14, 39, 71). Rapidtest kits such as the Rosco system (36), API Coryne (35), andMICRO-ID (1) have been developed recently by using theSA+/RE- reaction as a final stage in the identification of L.monocytogenes.

There have been some reports, however, of inconsistentreactions between L. monocytogenes and S. aureus. Stelma etal. (68) tested the virulence of a number of L. monocytogenesisolates by using immunocompromised mice. They found thatall virulent strains were SA+; however, one SA+ strain wasavirulent. Connor et al. (14) examined 218 cultures of L.monocytogenes for mouse pathogenicity. In that study, allpathogenic isolates were SA+; however, several nonpatho-genic, weakly hemolytic cultures were SA+. Van der Kellenand Lindsay (71) also compared virulent and avirulent L.monocytogenes strains and found an avirulent strain thatnevertheless gave a weak SA+ reaction. In contrast, Vazquez-Boland et al. (72) noted that the SA+ characteristic wasdifficult to interpret, leading to possible false-negative results.

In spite of the widespread belief in the RE- nature of L.monocytogenes, two studies by Skalka et al. (64, 65) showed aclear synergistic lysis between L. monocytogenes and eithercultures or cell-free culture fluid of R. equi, in agreement withthe earlier work by Fraser (28). These studies also linked anRE+ test result to virulence. Nakazawa and Nemoto (51)found that 25 of 25 strains of L. monocytogenes were RE'.Dominguez-Rodriguez et al. (22), working extensively with R.equi extracts, have developed a microplate technique for typingListeria spp. by using suspensions of horse RBC. They sug-gested that the apparent inability of L. monocytogenes to reactwith R. equi in some studies may be due to the use of anagar-based test, which is thought to be less sensitive than themicroplate method. A double CAMP test, which uses thereaction with both S. aureus and R. equi for identification of L.monocytogenes, was suggested by Smola (67). He noted theimportance of the RE' reaction and showed a correlation withvirulence. He also warned that strict standardization of R. equicultures was required in order to obtain a valid reaction.Schuchat et al. (60) have also emphasized the need forstandardized cultures of R. equi, and in agreement with this, ithas been observed that not all strains of R. equi produce thesynergistic factor (51). Since these detailed studies were done,

several other authors have reported synergism between L.monocytogenes and R. equi (46-48, 72).

CAMP REACTIONS OF OTHER LISTERL4 SPECIES

In addition to L. monocytogenes, the genus Listeria consistsof several species nonpathogenic for humans which may give apositive CAMP reaction: Listenia ivanovii, which is pathogenicfor animals (61) and was originally known as L. monocytogenesserotype 5 (62); Listeria innocua; and Listeria seeligen (61). Onblood agar, L. ivanovii produces bizonal clearing, with an innerclear zone and an outer, more diffuse zone of incompletehemolysis (63). The characteristic CAMP reaction of L. ivano-vii is SA-/RE+ (61), with the synergistic reaction with R. equibeing manifest in the zone of incomplete hemolysis (63, 65). L.ivanovii gives a typical "shovel-shaped" zone of clearing with R.equi, in contrast to the smaller, rounder zone observed be-tween L. monocytogenes and R. equi (65). Since these studieswere done, several other workers have confirmed the RE'reaction of L. ivanovii (4, 39, 68, 73). Some studies, however,have produced contradictory results. Using a microplate tech-nique, Dominguez-Rodriguez et al. (22) demonstrated that L.ivanovii hemolysis on horse RBC was enhanced by R. equi,Pseudomonas fluorescens, Acinetobacter calcoaceticus, and S.aureus.

L. innocua and L. seeligen are nonpathogenic for animalsand humans and are characterized by CAMP reactions ofSA-/RE- and SA+/RE-, respectively (61). A few contradic-tory results have been reported. Skalka et al. (66) noted thatwhile L. innocua is considered to be nonhemolytic (61), it gavea positive hemolysis on rabbit RBC which was not enhanced byR. equi. This apparent hemolysis was later attributed to lysis ofRBC by acid produced during growth of L. innocua (54). AnRE' reaction with L. seeligeri was also noted by Dominguez-Rodriguez et al. (22) with a microplate technique. L. seeligeristrains also gave variable results with R. equi on sheep bloodagar (72).

IDENTIFICATION OF CAMP FACTORS ANDMECHANISM OF ACTION

Most research efforts have been directed toward the devel-opment of a reliable, qualitative CAMP test for L. monocyto-genes which can be correlated with virulence; however, littleprogress has been made, in part because of poor standardiza-tion of test strains and assay conditions. The L. monocytogenesvirulence factors which may be involved in synergistic reactionshave not been adequately characterized, and few attempts toquantitate the method have been made. This is somewhatsurprising, since an improved assay technique can come onlyfrom a more thorough understanding of the mechanismsinvolved.

It has been known for some time that L. monocytogenesproduces a hemolysin, and the earlier work on this subject wasreviewed by Sword and Kingdon (70). This hemolysin was lateridentified as a pore-forming sulfhydryl-activated cytolysin (lis-teriolysin 0 [LLO]) similar to streptolysin 0 (29, 50), and ithas been directly implicated in virulence (15). The presenthypothesis is that LLO is required in order to lyse the hostvacuole, allowing L. monocytogenes to grow in the host cyto-plasm (55). Earlier studies (33, 37) showed that L. monocyto-genes produced a phospholipase or lecithinase activity, whichwas identified by its ability to react on egg yolk substrates.More recent work has ascribed phospholipase activity to twoenzymes: a phosphatidylinositol-specific phospholipase C (PI-PLC) (11, 41, 49, 53) and a phosphatidylcholine-specific phos-

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TABLE 1. Various reactions of L. monocytogenes mutantsa

Virulence factors CAMP reaction with: Reaction on blood agar:strainb -_ _ _ _ _ __ _ _ ___-_ _ _ _ _

LLOC PC-PLC PI-PLC S. aureus R. equi Without COBd With COB

Group ABUG 13 154.0 - + + + +BUG 206 192.0 + - + + + +++LUT 12 252.0 - + + + + +++

Group BMl 5.7 + + + - - +M3 5.0 + + + + - - +Hly- 4.2 ++ + + - - +

Group CM12 5.5 - - - - - +SLCC 5764M 21.0 - _ _ (+)e (+) +ATCC 43248 17.0 - - - (+) (+) +

a Reproduced from reference 47 with the permission of Blackwell Scientific Publications Ltd.b Details of sources of strains are given in reference 47.cLLO activity is expressed in hemolytic units per milliliter of cell-free culture fluid (45).d COB, cholesterol oxidase from B. brevi.e (+), weak positive reaction.

pholipase C (PC-PLC) (30, 34, 69). The PI-PLC has beenimplicated in virulence (11, 41, 49); however, its role has yet tobe determined. The PC-PLC is also required for virulence andmay play a role in cell-to-cell spread (74).The S. aureus factor involved in synergistic lysis reactions

(termed beta-toxin) has been identified as a PLC (EC 3.1.4.3)and specifically as a sphingomyelinase (20), which cleaves thecholine phosphate residue from membrane sphingomyelin,leaving the ceramide moiety in the membrane (6). The result-ing "sensitized" membranes may be subsequently attacked byother factors, such as the Streptococcus agalactiae CAMPfactor (6) or the C. renale renalin (5). Bernheimer et al. (7)found that a phospholipase D (phosphatidylcholine phos-phatidohydrolase [EC 3.1.4.4]) produced by C. ovis was alsoable to sensitize the RBC membranes by cleaving choline andleaving ceramide phosphate within the membrane. Theseworkers also showed that subsequent digestion of the sensi-tized membrane was effected by a PLC produced by R equi.Further work by this group revealed an additional mechanisminvolving digestion of cholesterol in previously sensitized mem-branes by a cholesterol oxidase (EC 1.1.3.6) produced by R.equi (43). Linder has reviewed the cooperative and antagonis-tic actions of bacterial proteins on mammalian membranes(42).

It is worthy of note that the only known mechanism ofsynergistic lysis of mammalian cells involves a sensitizationstep as described above. It must be emphasized that thesensitizing reaction is sequential; digestion with sphingomyeli-nase is an irreversible reaction, which must take place prior tocomplete lysis with synergistic factors.

It has been noted above that R equi produces two lyticfactors (a PLC and cholesterol oxidase) which may reactsynergistically with S. aureus. The first evidence for the involve-ment of R equi PLC in the synergistic reaction with L.monocytogenes was obtained by Nakazawa and Nemoto (51),who found that 22 strains ofR equi giving a positive egg yolkreaction also interacted synergistically with L. monocytogenes.Skalka et al. (65) suggested that synergism resulted from theproduction of PLC by both L. monocytogenes and R equi.Strangely, this group also proposed that LLO was the L.monocytogenes factor involved in the R. equi reaction (64).Other workers have suggested that either LLO (22, 67) or PLC(22) was the L. monocytogenes factor involved in the CAMPreaction with R equi.

Only a few attempts to identify the L. monocytogenes factorwhich reacts with S. aureus have been made. Datta et al. (17)developed a DNA probe for LLO and noted that strains of L.monocytogenes which were negative to the probe also gave anSA- reaction, suggesting that LLO is involved in the reactionwith S. aureus. These findings were supported by Connor et al.(14), who also noted weak S. aureus reactions with LLOprobe-negative strains of L. monocytogenes. Schonberg (58)also reported that nonhemolytic strains of L. monocytogenesdid not react with S. aureus.The first serious attempts to identify the L. monocytogenes

CAMP factors were by McKellar (46, 47), and this worker wasthe first to suggest that the S. aureus- and R. equi-L. monocy-togenes reactions involved different L. monocytogenes factors.In the first of these studies, the reaction between S. aureus andL. monocytogenes was found to be similar to that between S.aureus and R. equi; i.e., sensitization of the RBC membrane byS. aureus sphingomyelinase was required prior to completelysis with either L. monocytogenes or R equi. This is inagreement with the observations that RBC sensitized with C.ovis phospholipase D were further lysed byR equi (7) or by L.monocytogenes (48). In contrast, the L. monocytogenes-R. equireaction was not sequential; both lytic factors were requiredsimultaneously (46), suggesting that the L. monocytogenesfactor in this synergistic reaction was not a sphingomyelinase.Further, cholesterol oxidase of Brevibacterium brevi reactedsynergistically with either S. aureus or L. monocytogenes, whilethe cholesterol oxidase of P. fluorescens reacted only with L.monocytogenes, providing further evidence for the dissimilarityof the L. monocytogenes and S. aureus factors.The second study focused on the use of L. monocytogenes

mutants deficient in the production of extracellular lytic factors(47). Mutants lacking LLO were RE- while still retaining theirSA+ characteristic, suggesting that LLO is responsible for thereaction with R equi (Table 1). Mutants deficient in theproduction of either PT- or PC-PLC were SA+/RE+; loss ofsynergism with S. aureus was observed only with mutantslacking both PLCs. Thus, evidence has been presented toimplicate the PLCs of L. monocytogenes in the S. aureusreaction and LLO in the R. equi reaction.The actual mechanisms of the S. aureus- and R. equi-L.

monocytogenes reactions are unknown. In the S. aureus-L.monocytogenes reaction, it is likely that one of the PLCs fromL. monocytogenes digests phosphatidylcholine or ceramide in

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the RBC membrane after removal of the choline head groupsby the sphingomyelinase of S. aureus (47). The mechanism ofthe L. monocytogenes-R. equi reaction is less clear. It has beensuggested that limited attachment of LLO to the cell mem-brane allows entry of R. equi cholesterol oxidase to sensitivesites inside the membrane (47). This hypothesis is consistentwith previous observations; however, no direct evidence forthis interaction has been obtained to date.The factors involved in the synergistic lysis of RBC by L.

ivanovii are better understood than those for L. monocytogenes.Skalka and Smola (63) compared the beta-toxin (sphingomy-elinase) of S. aureus with the so-called "hemolysin" of L.ivanovii. They found that these two substances had similarcharacteristics: synergistic lysis of RBC with factors producedby other microorganisms, "hot-cold effect" (lysis of RBC uponcooling to 10°C [42]), and inhibition of synergistic lysis by thephospholipase D of C. ovis. A similar comparison between S.aureus and L. ivanovii was made by Brzin et al. (10), whosuggested that this phenomenon be used as a diagnostic test forL. ivanovii. Mencikova (48) confirmed the inhibition of L.ivanovii synergistic lysis by C. ovis and provided biochemicalevidence that the L. ivanovii factor was a PLC. Kreft et al. (38)characterized the L. ivanovii factors and identified sphingomy-elinase and lecithinase activities, which were later shown to bedifferent enzymes by using mutants (39). They also attributedthe second zone of incomplete hemolysis to the combinedaction of these phospholipases (38). These phospholipaseactivities were further characterized by Barclay et al. (2, 3).Vazquez-Boland et al. (73) purified the LLO and sphingo-mylelinase activities of L. ivanovii and implicated the sphingo-myelinase in the L. ivanovii-R. equi synergistic reaction.

ROLE OF BLOOD TYPE

One of the factors complicating the interpretation of thenumerous studies on the CAMP test is the many differentsources of RBC used. While ruminant (primarily sheep) bloodis favored, rat, pig, horse, guinea pig, and human bloodsamples have been used.The advantages of ruminant blood were first noted by

Christie et al. (13) during the development of the originalCAMP test. Fraser (28) noted that rabbit blood supportedmost synergistic reactions (including L. monocytogenes-R.equi); moreover, rabbit blood was required for the reactionbetween L. monocytogenes and either C. renale or C. ovis. Morerecent studies suggest that other blood types also support theCAMP reaction. Skalka and Smola (63) found that the S.aureus- or L. ivanovii-L. monocytogenes reaction was apparenton sheep, rabbit, horse, or pig blood. Skalka et al. (64) werefurther able to differentiate strains of L. monocytogenes on thebasis of their synergistic reactions with R. equi on sheep, horse,and human blood. Van der Kellen and Lindsay (71) comparedthe hemolytic activities of virulent and avirulent L. monocyto-getnes strains on various blood types. They found that theavirulent L. monocytogenes strain lysed human, rabbit, pig, andchicken blood, while sheep and horse blood gave no activity.The avirulent strain gave a weak response with S. aureus (71),suggesting that it may be deficient in phospholipase activity. Incontrast, the virulent L. monocytogenes strain was active onhuman, pig, and sheep blood.

It is very difficult to explain the divergent observations on theinfluence of RBC species on synergistic lysis in terms of themembrane composition. This may be attributed in part to thesubjective nature of many of the qualitative observations andto the use of poorly defined strains. Van der Kellen andLindsay (71) attributed varied hemolytic responses to differ-

ences in cholesterol content of the RBC membranes. This viewis supported by Linder and Bernheimer (44), who reportedthat human RBC with an increased content of membranecholesterol were more sensitive than normal cells to agentswhich interact with membrane sterol. Nelson (52) has noted,however, that most RBC species, including equine, rabbit, andruminant RBC, have roughly 26% of their total membranelipid as cholesterol. It is perhaps easier to explain the prefer-ence of most workers for ruminant RBC. A large proportion(>45%) of the phospholipid content of the ruminant RBCmembrane is composed of sphingomyelin, whereas othersources have 15 to 25% (52). As a result, synergistic lysis withS. aureus or C. ovis is favored, and a stronger, more consistentresponse is obtained.

ALTERNATIVES TO THE TRADITIONAL CAMP TEST

There have been many reports of difficulties in standardizingthe CAMP test for L. monocytogenes, which have been attrib-uted to the use of an essentially qualitative agar diffusion test.Coupled with the reported variability in production of LLOand other virulence factors by L. monocytogenes, the agar platemethod may have severe limitations. There have been severalattempts to improve the detection of hemolysis, based onenhancement of the CAMP reactions or on enhancement ofhemolysis in the absence of a synergistic factor.

Fraser (28) reported an enhancement of the S. aureus-L.monocytogenes reaction when cell-free culture fluids of the twostrains were combined. He also noted some difficulties inobtaining a suitable reaction between S. aureus and R. equiwhen the R. equi extract was prepared in broth culture. Theliquid culture method was improved by Dominguez-Rodriguezet al. (22), who developed a microplate technique. In thismethod, horse RBC were treated with crude exosubstancesproduced by S. aureus or R. equi and then exposed to dilutionsof L. monocytogenes cells in microplate wells. Dilutions causingcomplete lysis of the RBC were calculated, allowing quantita-tion of the L. monocytogenes factors. This method has theadditional advantage of being suitable for automation.Some attempts have been made to improve the more

traditional CAMP agar test. Skalka et al. (64, 65) haveproposed the use of blood agar impregnated with crudeextracts of R. equi. While this method is still qualitative, the useof standardized extracts would improve the reliability of theassay. McKellar (47) suggested that purified cholesterol oxi-dase may be used in place of extracts to eliminate the need forstandardized R. equi cultures. Filter disks impregnated withbeta-toxin were used with the agar assay by Connor et al. (14).There have also been some attempts to enhance hemolysis

on blood agar to avoid the need to include synergistic culturesor factors. Blanco et al. (9) and Dominguez-Rodriguez et al.(21) have used thin layers of blood agar overlaid on the surfaceof selective agar. Cassiday et al. (12) have suggested the use ofreplica plating onto blood agar. Improvements may beachieved by the use of more appropriate types of blood;Schuch et al. (59) substituted guinea pig blood for sheep bloodand noted improved hemolysis in an agar plate assay. Telluritehas also been used to enhance the hemolytic reaction of L.monocytogenes (25), in both liquid and agar assays. In thatstudy, mutants deficient in LLO activity did not give enhancedhemolysis with tellurite, whereas PLC- mutants responded totellurite. The authors suggested that tellurite stimulates LLOactivity; however, a recent study suggests that tellurite may actdirectly on the RBC membrane (18).

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CONCLUSIONS AND FUTURE DIRECTIONS

The evidence summarized in this review is consistent withthe hypothesis that the L. monocytogenes factors responsiblefor synergistic lysis of RBC with S. aureus and R. equi are PLCand LLO, respectively. Direct evidence for these mechanismsis lacking, however, and will require further studies usingpurified virulence factors. While the S. aureus factor (sphingo-myelinase) has been well characterized, the R. equi factor(s)involved in the reaction with L. monocytogenes has yet to beconclusively identified. McKellar (47) has implicated the R.equi cholesterol oxidase; however, the role of the R equi PLChas yet to be fully elucidated. Further research is warranted; inparticular, a more thorough analysis of the influence of RBCphospholipid and cholesterol composition on synergistic lysisshould shed some light on the mechanism of the CAMPreaction.The evidence for a direct link between L. monocytogenes

virulence and the CAMP reaction is still unconvincing. AListeria isolate giving a positive CAMP reaction with S. aureusand R equi must be considered a presumptive L. monocyto-genes isolate; however, the CAMP test is not sufficient toindicate whether the isolate is virulent. There have been someobservations of false-positive reactions with S. aureus (i.e.,avirulent L. monocytogenes), but this is consistent with thehypothesis that the S. aureus reaction may be attributed to twoL. monocytogenes PLCs. The observations that cultures lackingLLO activity or which are negative to DNA probes for LLOcan be SA+ have confused some workers but are consistentwith the PLC mechanism. False-positive reactions may also beattributed to reaction of avirulent species (e.g., an SA+reaction with L. seeligeri or RE' with L. ivanovii). L. monocy-togenes is the only Listeria species which is positive with both S.aureus and R equi, however. False-negative reactions (i.e.,virulent L. monocytogenes not giving an S. aureus or R. equireaction) may present a more serious problem. False-negativereactions with S. aureus may result from difficulty in readingthe reaction on blood agar plates, and while false-negativereactions with R equi have been reported, most of theseprobably resulted from poor standardization of test strains.

Thus, an SA+/RE+ reaction is necessary but not sufficientfor the identification of virulent L. monocytogenes; further testsare necessary in order to clearly demonstrate virulence. Ani-mal pathogenicity tests are currently used, but these appear tohave some limitations (14, 68, 71).A new test based on the absence of arylamidase in L.

monocytogenes has been suggested as a method to differentiateL. monocytogenes from L. innocua (8). This test may wellreplace the CAMP test as a definitive test for L. monocyto-genes; however, tests based on enzymes unrelated to virulencemay not be acceptable.

REFERENCES1. Bannerman, E., M. N. Yersin, and J. Bille. 1992. Evaluation of the

Organon-Teknika MICRO-ID LISTERIA System. Appl. Environ.Microbiol. 58:2011-2015.

2. Barclay, R., D. R. Threlfall, and I. Leighton. 1989. Haemolysinsand extracellular enzymes of Listeria monocytogenes and L. ivano-vii. J. Med. Microbiol. 30:111-118.

3. Barclay, R., D. R. Threlfall, and I. Leighton. 1989. Separation andproperties of the haemolysins and extracellular enzymes of Listeriamonocytogenes and L. ivanovii. J. Med. Microbiol. 30:119-127.

4. Benedict, R. C. 1990. Listeria monocytogenes: physiology andmetabolism, p. 13-24. In A. J. Miller, J. L. Smith, and G. A.Somkuti (ed.), Topics in industrial microbiology. Foodborne list-eriosis. Elsevier, Amsterdam.

5. Bernheimer, A. W., and L. S. Avigad. 1982. Mechanism of hemo-

lysis by renalin, a CAMP-like protein from Corynebactenium renale.Infect. Immun. 36:1253-1256.

6. Bernheimer, A. W., R Linder, and L. S. Avigad. 1979. Nature andmechanism of action of the CAMP protein of group B strepto-cocci. Infect. Immun. 23:838-844.

7. Bernheimer, A. W., R. Linder, and L S. Avigad. 1980. Stepwisedegradation of membrane sphingomyelin by corynebacterial phos-pholipases. Infect. Immun. 29:123-131.

8. Bille, J., B. Catimel, E. Bannerman, C. Jacquet, M. N. Yersin, I.Caniaux, D. Monget, and J. Rocourt. 1992. API Listeria, a new andpromising one-day system to identify Listeria isolates. Appl. Envi-ron. Microbiol. 58:1857-1860.

9. Blanco, M., J. F. Fernandez-Garayzabal, L. Dominguez, V. Bri-ones, J. A. Vazquez-Boland, J. L. Blanco, J. A. Garcia, and G.Suarez. 1989. A technique for the direct identification of haemo-lytic-pathogenic listeria on selective plati:.g media. Lett. Appl.Microbiol. 9:125-128.

10. Brzin, B., N. Kuhar, B. Naverznik, and A. Vadnjal. 1990. Func-tional similarity of Listeria ivanovii and Staphylococcus aureus inCAMP test. Zentralbl. Bakteriol. Int. J. Med. Microbiol. 273:179-183.

11. Camilli, A., H. Goldfine, and D. A. Portnoy. 1991. Listeria mono-cytogenes mutants lacking phosphatidylinositol-specific phospho-lipase-C are avirulent. J. Exp. Med. 173:751-754.

12. Cassiday, P. K., L. M. Graves, and B. Swaminathan. 1990. Replicaplating of colonies from Listeria-selective agars to blood agar toimprove the isolation of Listeria monocytogenes from foods. Appl.Environ. Microbiol. 56:2274-2275.

13. Christie, R, N. E. Atkins, and E. Munch-Petersen. 1944. A noteon a lytic phenomenon shown by group B streptococci. Aust. J.Exp. Biol. Med. Sci. 22:197-200.

14. Conner, D. E., V. N. Scott, S. S. Sumner, and D. T. Bernard. 1989.Pathogenicity of foodborne, environmental and clinical isolates ofListeria monocytogenes in mice. J. Food Sci. 54:1553-1556.

15. Cossart, P., M. F. Vicente, J. Mengaud, F. Baquero, J. C.Perez-Diaz, and P. Berche. 1989. Listeriolysin 0 is essential forvirulence of Listeria monocytogenes: direct evidence obtained bygene complementation. Infect. Immun. 57:3629-3636.

16. Croize, J., J. Arvieux, P. Berche, and M. G. Colomb. 1993.Activation of the human complement alternative pathway byListeria monocytogenes: evidence for direct binding and proteolysisof the C3 component on bacteria. Infect. Immun. 61:5134-5139.

17. Datta, A. R, B. A. Wentz, and W. E. Hill. 1988. Identification andenumeration of beta-hemolytic Listeria monocytogenes in naturallycontaminated dairy products. J. Assoc. Off. Anal. Chem. 71:673-675.

18. Deuticke, B., P. Lutkemeier, and B. Poser. 1992. Tellurite-induceddamage of the erythrocyte membrane-manifestations and mech-anisms. Biochim. Biophys. Acta 1109:97-107.

19. Dillon, R M., and T. R Patel. 1992. Listeria in seafoods-areview. J. Food Prot. 55:1009-1015.

20. Doery, H. M., B. J. Magnusson, J. Gulasekharam, and J. E.Pearson. 1965. The properties of phospholipase enzymes in staph-ylococcal toxins. J. Gen. Microbiol. 40:283-296.

21. Dominguez, L., J. F. Fernandez-Garayzabal, M. D. Blanco, V.Briones, J. A. Vazquez-Boland, J. Blanco, and G. Suarez. 1990.Overlay technique for direct detection and identification of hae-molytic Listeria on selective plating medium-comparison of Smedia. Z. Lebensm. Unters. Forsch. 191:16-19.

22. Dominguez-Rodriguez, L., J. A. Vazquez-Boland, J. F. Fernandez-Garayzabal, P. Echalecu-Tranchant, E. Gomez-Lucia, E. F. Ro-driguez-Ferri, and G. Suarez-Fernandez. 1986. Microplate tech-nique to determine hemolytic activity for routine typing of Listeriastrains. J. Clin. Microbiol. 24:99-103.

23. Donnelly, C. W., R E. Brackett, S. Doores, W. H. Lee, and J.Lovett. 1992. Listeria, p. 637-663. In C. Vanderzant and D. F.Splitstoesser (ed.), Compendium of methods for the microbiolog-ical examination of foods, 3rd ed. American Public Health Asso-ciation, Washington, D.C.

24. Farber, J. M., and P. I. Peterkin. 1991. Listeria monocytogenes, afood-borne pathogen. Microbiol. Rev. 55:476-511.

25. Fernandez-Garayzabal, J. F., C. Delgado, M. Blanco, J. A.Vazquez-Boland, V. Briones, G. Suarez, and L. Dominguez. 1992.

VOL. 60, 1994

on Novem

ber 24, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

APPL. ENVIRON. MICROBIOL.

Role of potassium tellurite and brain heart infusion in expressionof the hemolytic phenotype of Listena spp. on agar plates. Appl.Environ. Microbiol. 58:434-438.

26. Flowers, R. S., W. Andrews, C. W. Donnelly, and E. Koenig. 1992.Pathogens in milk and milk products, p. 103-212. In R. T. Marshall(ed.), Standard methods for the examination of dairy products,16th ed. American Public Health Association, Washington, D.C.

27. Fraser, G. 1962. A plate method for the rapid identification ofListeria (Erysipelothrix) monocytogenes. Vet. Rec. 74:50.

28. Fraser, G. 1964. The effect on animal erythrocytes of combinationsof diffusible substances produced by bacteria. J. Pathol. Bacteriol.88:43-53.

29. Geoffroy, C., J.-L. Gaillard, J. E. Alouf, and P. Berche. 1987.Purification, characterization and toxicity of the sulfhydryl-acti-vated hemolysin listeriolysin 0 from Listeria monocytogenes. In-fect. Immun. 55:1641-1646.

30. Geoffroy, C., J. Raveneau, J.-L. Beretti, A. Lecroisey, J. A.Vazquez-Boland, J. E. Alouf, and P. Berche. 1991. Purification andcharacterization of an extracellular 29-kilodalton phospholipase Cfrom Listeria monocytogenes. Infect. Immun. 59:2382-2388.

31. Groves, R. D., and H. J. Welshimer. 1977. Separation of patho-genic from apathogenic Listeria monocytogenes by three in vitroreactions. J. Clin. Microbiol. 5:559-563.

32. Jemmi, T. 1993. Listeria monocytogenes in smoked fish-an over-view. Arch. Lebensmittelhyg. 44:10-13.

33. Jenkins, E. M., and B. B. Watson. 1971. Extracellular antigensfrom Listeria monocytogenes. I. Purification and resolution ofhemolytic and lipolytic antigens from culture filtrates of Listenamonocytogenes. Infect. Immun. 3:589-594.

34. Kathariou, S., L. Pine, V. George, G. M. Carlone, and B. P.Holloway. 1990. Nonhemolytic Listeria monocytogenes mutantsthat are also noninvasive for mammalian cells in culture: evidencefor coordinate regulation of virulence. Infect. Immun. 58:3988-3995.

35. Kerr, K. G., P. M. Hawkey, and R. W. Lacey. 1993. Evaluation ofthe API Coryne system for identification of Listeria species. J. Clin.Microbiol. 31:749-750.

36. Kerr, K. G., N. A. Rotowa, P. M. Hawkey, and R. W. Lacey. 1991.Evaluation of the Rosco system for the identification of Listenaspecies. J. Med. Microbiol. 35:193-196.

37. Khan, M. A., A. Seaman, and M. Woodbine. 1973. Listeria mono-cytogenes-haemolysin:lecithinase. Zentralbl. Bakteriol. Hyg. Abt.1 Orig. A 225:66-79.

38. Kreft, J., D. Funke, A. Haas, F. Lottspeich, and W. Goebel. 1989.Production, purification and characterization of hemolysins fromListeria ivanovii and Listeria monocytogenes Sv4b. FEMS. Micro-biol. Lett. 48:197-202.

39. Kreft, J., D. Funke, R. Schlesinger, F. Lottspeich, and W. Goebel.1989. Purification and characterization of cytolysins from Listeriamonocytogenes serovar 4b and Listeria ivanovii. Acta Microbiol.Hung. 36:189-192.

40. Kuhn, M., and W. Goebel. 1994. Induction of cytokines in phago-cytic mammalian cells infected with virulent and avirulent Listeriastrains. Infect. Immun. 62:348-356.

41. Leimeister-Wachter, M., E. Domann, and T. Chakraborty. 1991.Detection of a gene encoding a phosphatidylinositol-specific phos-pholipase-C that is coordinately expressed with listeriolysin inListenia monocytogenes. Mol. Microbiol. 5:361-366.

42. Linder, R. 1984. Alteration of mammalian membranes by thecooperative and antagonistic actions of bacterial proteins. Bio-chim. Biophys. Acta 779:423-435.

43. Linder, R., and A. W. Bernheimer. 1982. Enzymatic oxidation ofmembrane cholesterol in relation to lysis of sheep erythrocytes bycorynebacterial enzymes. Arch. Biochem. Biophys. 213:395-404.

44. Linder, R., and A. W. Bernheimer. 1984. Action of bacterialcytotoxins on normal mammalian cells and cells with alteredmembrane lipid composition. Toxicon 22:641-651.

45. McKellar, R. C. 1992. Effect of reduced pH on secretion, stabilityand activity of Listeria monocytogenes listeriolysin 0. J. FoodSafety 12:283-293.

46. McKellar, R. C. 1993. Novel mechanism for the CAMP reactionbetween Listena monocytogenes and Corynebacterium equi. Int. J.Food Microbiol. 18:77-82.

47. McKellar, R. C. 1994. Identification of the virulence factorsinvolved in the CAMP reaction between Listeria monocytogenesand Corynebacterium equi. Lett. Appl. Microbiol. 18:79-81.

48. Mencikova, E. 1989. Phospholipase C in Listeria. Acta Microbiol.Hung. 36:321-325.

49. Mengaud, J., C. Braunbreton, and P. Cossart. 1991. Identificationof phosphatidylinositol-specific phospholipase-C activity in Liste-na monocytogenes-a novel type of virulence factor. Mol. Micro-biol. 5:367-372.

50. Mengaud, J., J. Chenevert, C. Geoffroy, J.-L. Gaillard, and P.Cossart. 1987. Identification of the structural gene encoding theSH-activated hemolysin of Listeria monocytogenes: listeriolysin 0is homologous to streptolysin 0 and pneumolysin. Infect. Immun.55:3225-3227.

51. Nakazawa, M., and H. Nemoto. 1980. Synergistic hemolysis phe-nomenon of Listenia monocytogenes and Corynebactenium equi.Jpn. J. Vet. Sci. 42:603.

52. Nelson, G. J. 1967. Lipid composition of erythrocytes in variousmammalian species. Biochim. Biophys. Acta 144:221-232.

53. Notermans, S. H. W., J. Dufrenne, M. Leimeister-Wachter, E.Domann, and T. Chakraborty. 1991. Phosphatidylinositol-specificphospholipase C activity as a marker to distinguish betweenpathogenic and nonpathogenic Listeria species. Appl. Environ.Microbiol. 57:2666-2670.

54. Pongratz, G., and H. P. R. Seeliger. 1984. Hemolytic effects on

sheep erythrocytes by Listeria innocua. Zentralbl. Bakteriol. Mik-robiol. Hyg. Abt. 1 Orig. B 257:296-307.

55. Portnoy, D. A., T. Chakraborty, W. Goebel, and P. Cossart. 1992.Molecular determinants of Listeria monocytogenes pathogenesis.Infect. Immun. 60:1263-1267.

56. Roberts, T., and R. Pinner. 1990. Economic impact of diseasecaused by Listeria monocytogenes, p. 137-149. In A. J. Miller, J. L.Smith, and G. A. Somkuti (ed.), Topics in industrial microbiology.Foodborne listeriosis. Elsevier, Amsterdam.

57. Ryser, E. T., and E. H. Marth. 1991. Listeria, listeriosis, and foodsafety. Marcel Dekker, Inc., New York.

58. Schonberg, A. 1989. Method to determine virulence of Listeriastrains. Int. J. Food Microbiol. 8:281-284.

59. Schuch, D. M. T., J. Moore, R. H. Madden, and W. E. Espie. 1992.Haemolytic reaction of Listeria monocytogenes on bilayer columbiaagar plates with defibrinated guinea-pig blood. Lett. Appl. Micro-biol. 15:78-79.

60. Schuchat, A., B. Swaminathan, and C. V. Broome. 1991. Listenamonocytogenes CAMP reaction. Clin. Microbiol. Rev. 4:396.

61. Seeliger, H. P. R., and D. Jones. 1986. Genus Listenia, p. 1235-1245. In P. H. A. Sneath, N. S. Mair, M. E. Sharpe, and J. G. Holt(ed.), Bergey's manual of systematic bacteriology, vol. 2. TheWilliams & Wilkins Co., Baltimore.

62. Seeliger, H. P. R., and H. J. Welshimer. 1974. Genus Listeria, p.593-596. In R. E. Buchanan and N. E. Gibbons (ed.), Bergey'smanual of determinative bacteriology, 8th ed. The Williams &Wilkins Co., Baltimore.

63. Skalka, B., and J. Smola. 1982. Hemolytic properties of exosub-stances of serovar 5 Listeria monocytogenes compared with betatoxin of Staphylococcus aureus. Zentralbl. Bakteriol. Hyg. Abt. 1Orig. A 252:17-25.

64. Skalka, B., J. Smola, and K. Elischerova. 1982. Different haemo-lytic activities of Listeria monocytogenes strains determined onerythrocytes of various sources and exploiting the synergism ofequi-factor. Zentralbl. Veterinaermed. Reihe B 29:642-649.

65. Skalka, B., J. Smola, and K. Elischerova. 1982. Routine test for invitro differentiation of pathogenic and apathogenic Listenia mono-cytogenes strains. J. Clin. Microbiol. 15:503-507.

66. Skalka, B., J. Smola, and K. Elischerova. 1983. Hemolytic phe-nomenons under the cultivation of Listeria innocua. Zentralbl.Bakteriol. Hyg. Abt. 1 Orig. A 253:559-565.

67. Smola, J. 1989. Possibilities of differentiation of listerial hemo-lysins by synergistic hemolytic reactions (CAMP reactions). Int. J.Food Microbiol. 8:265-267.

68. Stelma, G. N., Jr., A. L. Reyes, J. T. Peeler, D. W. Francis, J. M.Hunt, P. L. Spaulding, C. H. Johnson, and J. Lovett. 1987.Pathogenicity test for Listeria monocytogenes using immunocom-promised mice. J. Clin. Microbiol. 25:2085.

4224 MINIREVIEW

on Novem

ber 24, 2020 by guesthttp://aem

.asm.org/

Dow

nloaded from

MINIREVIEW 4225

69. Sun, A. N., A. Camilli, and D. A. Portnoy. 1990. Isolation ofListeria monocytogenes small-plaque mutants defective for intra-cellular growth and cell-to-cell spread. Infect. Immun. 58:3770-3778.

70. Sword, C. P., and G. C. Kingdon. 1971. Listenia monocytogenestoxin, p. 357-377. In S. Kadis, T. C. Montie, and S. J. Ajl (ed.),Microbial toxins. Academic Press, New York.

71. Van der Kelen, D., and J. A. Lindsay. 1990. Differential hemolyticresponse of Listeria monocytogenes strains on various blood agars.J. Food Safety 11:9-12.

72. Vazquez-Boland, J. A., L. Dominguez, J. F. Fernandez, E. F.Rodriguez-Ferri, V. Briones, M. Blanco, and G. Suarez. 1990.Revision of the validity of CAMP tests for Listeria identification-proposal of an alternative method for the determination ofhaemolytic activity by Listeria strains. Acta Microbiol. Hung. 37:201-206.

73. Vazquez-Boland, J. A., L. Dominguez, E. F. Rodriguez-Ferri, and

G. Suarez. 1989. Purification and characterization of two Listeriaivanovii cytolysins, a sphingomyelinase C and a thiol-activatedtoxin (ivanolysin 0). Infect. Immun. 57:3928-3935.

74. Vazquez-Boland, J. A., C. Kocks, S. Dramsi, H. Ohayon, C.Geoffroy, J. Mengaud, and P. Cossart 1992. Nucleotide sequenceof the lecithinase operon of Listeria monocytogenes and possiblerole of lecithinase in cell-to-cell spread. Infect. Immun. 60:219-230.

75. Warburton, D. W., J. M. Farber, and T. Babiuk 1992. Isolation ofListeria monocytogenes from all food and environmental samples.MFHPB-30. HPB methods of microbial analysis of food, vol. 2.Polyscience Publications Inc., Montreal.

76. Yamamoto, S., F. Russ, H. C. Teixeira, P. Conradt, and S. H. E.Kaufmann. 1993. Listeria monocytogenes-induced gamma inter-feron secretion by intestinal intraepithelial gamma/delta T lym-phocytes. Infect. Immun. 61:2154-2161.

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