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Differential Diagnosis Between Streptococcus Agalactiae and Listeria Monocytogenes in the Clinical Laboratory

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Differential Diagnosis Between Streptococcus Agalactiae and Listeria Monocytogenes in the Clinical Laboratory ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 7, No. 3 Copyright © 1977, Institute for Clinical Science Differential Diagnosis between Streptococcus Agalactiae and Listeria Monocytogenes in the Clinical Laboratory CHRISTINE KONTNICK, M.T., ALEXANDER von GRAEVENITZ, M.D., and VINCENT PISCITELLI, M.T. Clinical Microbiology Laboratories, Yale-New Haven Hospital, and Department of Laboratory Medicine, Yale University School of Medicine, New Haven, CT 06504 ABSTRACT Streptococci of the group B (S. agalactiae) and Listeria monocytogenes resemble each other in many morphological and biochemical characteris­ tics. Ten beta-hemolytic strains of each species were subjected to 26 tests commonly and easily performed in the clinical laboratory. Macroscopic and microscopic morphology on solid media showed differences only in the size of the colonies and in the length of the individual organisms. Among many other tests, hippurate hydrolysis and the CAMP reaction were pos­ itive in both species. In the presence of these two reactions, a negative catalase test and chaining in broth would make a presumptive diagnosis of S. agalactiae, while motility at 25 C, the presence of the Henry effect, and resistance to furadantin would be indicative of L. monocytogenes. Introduction in Gram-stained smears; (3) a negative bacitracin test and (4) a positive test for The high incidence of Streptococcus either (a) hippurate hydrolysis, (b) the agalactiae (group B) in human speci­ CAMP reaction or (c) the formation of an mens, which has been recognized only in orange-red pigment. However, if one of the past decade, calls for a rapid pre­ these conditions for the diagnosis is not sumptive diagnosis of the species. Many met, differential diagnostic problems clinical laboratories, at least the small may arise with other Gram-positive or­ ones, prefer not to perform serological ganisms. This is illustrated by the follow­ tests (CIE, coagglutination) for the iden­ ing facts: tification of streptococci because of the expenses in equipment, time and money (1). A minority of S. agalactiae strains involved. For them, the presumptive is nonhemolytic on sheep or rabbit blood diagnosis of S. agalactiae usually rests agar even under anaerobic condi­ with the combination of: (1) beta- tions.18,45 On the other hand, colonies of hemolytic colonies on sheep or rabbit beta-hemolytic Gram-positive organisms blood agar; (2) appearance of streptococci like Corynebacterium haemolyticum, 2 7 0 K ON TN ICK, v o n GRAEVENITZ AND PISCITELLI Listeria monocytogenes and other beta- served in S. agalactiae but this charac­ hemolytic streptococci may closely re­ teristic is highly dependent on semble those of S. agalactiae. L. anaerobiosis and the medium used; Col­ monocytogenes colonies41 tend to be umbia agar being the optimal one.16,34 It smaller in size than those of S. agalac­ may be absent in 3 percent of the beta- tiae,6 but the hemolytic zone of both is hemolytic34 and in some of the nonhemo­ usually small. lytic strains.16 Some L. monocytogenes (2). Chain formation of streptococci is strains are also said to produce a yel­ seen best in Gram-stained smears from lowish pigment after several months of liquid media1 and may be poor in Gram incubation.42 C. poinsettiae, a motile stains from solid media. Thus, coccoid Corynebacterium, also forms an orange Gram-positive organisms on solid media pigment.10 may be streptococci, but may also be It is thus obvious that one Gram- young listeriae10 (which may even form positive rod, L. monocytogenes, shares short chains).41 In broth, however, S. many features with S. agalactiae. The agalactiae usually forms long chains.17 possibility of a misdiagnosis is further (3). The bacitracin test is positive in 4 enhanced by the fact that both species to 8 percent of S. agalactiae, streptococci tend to be isolated mainly from the cer­ of groups other than A, B, and D, S. bovis, vix, from blood, and from the spinal fluid S. viridans and in about 48 percent of S. (although group B wound infections are pneumoniae; however, it is negative in not uncommon). In fact, L. monocyto­ about 0.5 percent of S. pyogenes.15 genes tends to be most often misdiag­ (4a). The hippurate test is reported nosed as a streptococcus species.10,20,41 positive among Gram-positive bacteria The smear of a liquid sample or culture regularly in S. bovis and S. acidomin- would immediately differentiate the two, imusK in L. monocytogenes (but not in but if a Gram stain is done either from a other Listeria species),14,15* in certain specimen that contains other streptococ­ species of Lactobacillus39 and Coryne- ci from a plate, or if no Gram stain is done bacterium7 and in about 6.9 percent of at ail (as is often the case on cervical enterococcus strains.15 smears), a misdiagnosis may ensue. (4b). The CAMP test36 is positive in S. The present investigation was promp­ pyogenes (group A) under anaerobic con­ ted by a routine search for S. agalactiae ditions,11 but may yield equivocal results in cervical specimens in the course of under aerobic conditions.32 It has also which a strain of L. monocytogenes was been reported positive (aerobically) in 12 discovered. It was restricted to 26 com­ to 20 percent of group G, K, and L strep­ monly and easily performed tests, not in­ tococci,24 in certain Corynebacterium cluding macroscopic and microscopic species,19 and in virulent strains of L. morphology. Other tests did not appear monocytogenes.3,8,19,23 It may be nega­ feasible for routine purposes, as will be tive in S. agalactiae if the sheep blood pointed out in the discussion. used contains staphylococcal beta anti­ toxin and has not been washed,36 and may Materials and Methods also be negative in nonhemolytic strains Ten strains each of S. agalactiae and L. of group B.12,35 monocytogenes were examined. The S. (4c). Production of an orange-red pig­ agalactiae strains had been isolated from ment in streptococci has only been ob­ cervical specimens in this laboratory and were grouped by the Connecticut State * In the abstract of this paper,28 five of five L. monocytogenes strains were erroneously described Department of Health. Seven L. as “failing to hydrolyze hippurate.” monocytogenes strains had been isolated STREPTOCOCCUS AGALACTIAE VS. LISTERIA MONOCYTOGENES 2 7 1 TABLE I Tests and Methods Test Medium Method Positive Control Negative Control Arginine dihydrolase Decarboxylase Falkow1 Arizona hinshawii E. coli medium base* Bile-esculin (growth & Bile-esculin Swan1 S. faecalis E. coli esculin hydrolysis) agar* D a rl i ng 12CAMP reaction Tryptic soy agar Darling12CAMP S. agalactiae E. coli (aerobic) with 5% sheep blood* Catalase Tryptic soy broth* Broth1 E. coli 5. faecalis Citrate utilization Koser citrate Koser29 Klebsiella pneumoniae E. coli medium* Deoxyribonuclease DNase test agar von Graevenitz Serratia marcescens E. coli with methyl green* (24 & 48 hrs) Gelatin hydrolysis Photographic Lagodsky & S. marcescens E. coli X-ray strip J a n d a r d ^ Gluconate oxidation Tabletst Haynes1 Pseudomonas aeruginosa P. maltophilia Beta hemolysis Tryptic soy agar ~ S. pyogenes S. faecalis with 5% sheep blood* Henry effect Mueller-Hinton agar* Gray L. monocytogenes E. coli Hippurate hydrolysis - Hwang & Ederer S. agalactiae E. coli Hydrogen sulfide PathoTec strip§ Standard4 A. hinshawii E. coli Indole Trypticase*0 Ehrlich-Böhme1 E. coli Enterobacter aerogenes Lipase Spirit blue agar Hugo & S. marcescens E. coli with tributyrin* Beveridge2^ Lysine decarboxylase PathoTec strip§ Standard4 E. aerogenes Citrobacter diversus Malonate utilization Malonate broth* Leifson1 C. diversus E. coli Methyl red reaction MR-VP medium* Clark & Lubs1 E. coli K. pneumoniae Motility 25C Tryptic soy broth* Hanging drop1 E. coli K. pneumoniae Nitrate reducation Trypticase nitrate Standard1 E. coli S. faecalis brothw ONPG Tabletst Standard1 E. coli Salmonella sp. Ornithine decarboxylase PathoTec stripi Standard4 E. aerogenes Proteus vulgaris Oxidase - Ko va c s 1 P. aeruginosa E. coli Phenylalanine Urea-phenyalanine Ederer et al13 P. vulgaris E. coli deaminase medium* Mueller-Hinton agarw Kirby-Bauer1Antimicrobial E. coli ATCC 25922 - susceptibility Urea-phenyalanine Ederer et a l 1^Urease P. vulgaris E. coli medium* MR-VP medium* Coblentz1Voges-Proskauer K. pneumoniae E. coli reaction (37°C, 48 hrs) Voges-Proskauer PathoTec strip§ Standard4 K. pneumoniae E. coli *Difco Laboratories, Detroit, MI tKey Scientific Products, Los Angeles, CA §General Diagnostics Div., Warner-Lambert Company, Morris Plains, NJ UBBL, BioQuest, Cockeysville, MD from blood cultures here, and three were both Tryptic Soy Brothf and from blood received from the Bacteriology Labora­ agar after 24 hours of growth. In table I tory at the Massachusetts General Hospi­ are listed tests, methods and control tal, Boston. strains used. Incubation was at 37 for 24 Colonial morphology was observed on hours unless indicated otherwise, e.g., by blood agar (Tryptic Soy Agarf with 5 per­ the manufacturer of test strips or tablets. cent sheep blood) after incubation for 24 to 48 hours at 37° in 7.5 percent C02. Results Gram-stained smears were prepared from The colonial morphology of the strains t Difco Laboratories, Detroit, MI. was similar except for the larger diame- 2 7 2 K ON TN ICK, v o n GRAEVENITZ AND PISCITELLI TABLE I I Results (Positive Strains per Total Strains Tested) Results References S. agalactiae L. monocytogenes S. agalactiae L. monocytogenes 17 Arginine dihydrolase 10/10 0/10 3 ,44 15 ,17 ,27 27 Bile-esculin (growth & hydrolysis) 0/10 10/10
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