Nutritionally Variant Streptococci from Corneal Ulcers in Horses

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1 JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1984, p /84/ $02.00/0 Copyright 1984, American Society for Microbiology Vol. 20, No. 6 Nutritionally Variant Streptococci from Corneal Ulcers in Horses ROBERT HIGGINS,' ERNST L. BIBERSTEIN,2* AND SPENCER S. JANG3 Departement de Pathologie et Microbiologie, Faculte de Medecine Veterinaire, Universite de Montreal, Saint-Hyacinthe, Quebec, Canada J2S 7C61; and Department of Veterinary Microbiolofy and Immunology, School of Veterinary Medicine,2 and Microbiology Service, Veterinary Medical Teaching Hospital, University of California, Davis, California Received 1 June 1984/Accepted 20 August 1984 Of 24 isolates of nutritionally variant streptococci recovered from equine corneal ulcers, 22 were tested for growth requirements, physiological and biochemical reactions, and susceptibility to different antimicrobial agents. Satisfactory growth was obtained by supplementing blood agar and Todd-Hewitt broth with pyridoxal hydrochloride, and all of the media for the culture and the biochemical testing were supplemented with 0.002% of this substance. Biochemical patterns of 12 of the isolates resembled those of two viridans streptococcal species, Streptococcus intermedius and Streptococcus constellatus. Patterns of 10 isolates did not resemble those of any recognized viridans species. All of the isolates were inhibited by <0.25,ug of erythromycin per ml, c4,ug of chloramphenicol per ml, and c4,ug of gentamicin per ml, and all but two were inhibited by.0.1,ug of penicillin per ml. Nutritionally variant streptococci (NVS) are fastidiously growing viridans streptococci that have been recognized by their growth as satellite colonies around colonies of Staphylococcus spp. or several other bacterial species (3, 13). Other descriptive names have been assigned to this group of bacteria, including satelliting streptococci, thiol-dependent streptococci, vitamin B6-dependent streptococci, and symbiotic streptococci (9). Nutritional studies indicate that thiolcontaining compounds and various forms of vitamin B6 supply the required growth factors (9). The first isolation of NVS was reported in 1961, from human patients with subacute bacterial endocarditis (13). Since that time, numerous isolations have been reported, mostly from subacute bacterial endocarditis (3-5, 7, 9, 17, 19) but also from a pancreatic abscess (5), vaginal infections, otitis externa, wound infections, and saliva (14). The majority of the isolates were classified as Streptococcuis mitior (mitis) (3, 5, 9, 19), and the others were classified as Streptococcus morbillorum, Streptococcus sanguis II, Streptococcus salivarius, and Streptococcus anginosus-constellatus (9). In the summers of 1982 and 1983, a total of 24 isolates of satelliting streptococci were isolated by the Microbiology Service, Veterinary Medical Teaching Hospital, University of California at Davis. All of the isolates were found in horses, and 18 of the 24 isolates were recovered during a period of about 4 months, between July and October Interestingly, all 24 isolates were associated with corneal ulcers. Our study describes the physiological and biochemical properties of these organisms, their growth requirements, and their susceptibility to some antimicrobial substances. MATERIALS AND METHODS Strains. Four reference strains were kindly supplied by Richard R. Facklam, Clinical Bacteriology Branch, Centers for Disease Control, Atlanta, Ga. These are S. mitis SS-429, S. salivarius SS-262, S. intermedius SS-898 (Centers for Disease Control strain), and S. anginosus-constellatus SS * Corresponding author A total of 24 clinical isolates were recovered from the equine corneal swabs. The first isolates had been obtained only due to the incidental presence of colonies of contaminating staphylococci which acted as feeders, supplying the essential nutrients. Subsequently, the isolations were performed on 5% bovine blood agar with a single streak of Staphylococcus aureus overlaid on all equine-corneal-swab cultures. Plates were incubated at 37 C in candle jars. Satelliting colonies were subcultured onto chocolate agar with a streak of Staphylococcus aureus. The cultures were identified as NVS on the basis of satellitism, Gram stain, colonial morphology, and catalase reaction. They were then inoculated into skim milk tubes and stored at -70 C. Of the 24 isolates, 22 were successfully retrieved from storage. In total, 22 clinical isolates and four reference strains were studied. Growth requirements. The following media were tested for their ability to support the growth of five of the clinical isolates: unsupplemented blood agar (tryptose blood agar base [Difco Laboratories, Detroit, Mich.] with 5% bovine blood); unsupplemented chocolate agar; blood agar supplemented with 1.0% IsoVitaleX (BBL Microbiology Systems, Cockeysville, Md.); blood agar supplemented with 0.002% pyridoxal hydrochloride (Sigma Chemical Co., St. Louis, Mo.); unsupplemented tryptose broth (TB) and Todd-Hewitt broth (THB [Difco]); and TB and THB supplemented with 0.002% pyridoxal hydrochloride. Cultures were incubated in candle jars, and growth was estimated by a semiquantitative method described by Barry (1). The cultures were inoculated onto a small area, about the size of a quarter, and then streaked with a sterile bacteriological loop over the four overlapping quadrants of the plates. The amount of growth was scored from 1+ to 4+, by the quadrant in which five or more isolated colonies were observed. To test whether the growth of all of the 22 isolates could be enhanced by the presence of pyridoxal hydrochloride, onefourth-inch (0.635-cm) concentration disks (Difco) were inpregnated with 0.25 ml of a solution containing 100 pug of pyridoxal hydrochloride per ml of sterile distilled water. A wet disk was placed in the center of each blood agar plate inoculated with an isolate. Incubation was carried out as

2 VOL. 20, 1984 NUTRITIONALLY VARIANT STREPTOCOCCI IN HORSES 1131 TABLE 1. Evaluation of the growth of five NVS isolates in different media" BA with.002% 0.002% Pyri- Time BA or BA with 1% pyridoxal hydro- doxal hydro- (h) CA IsoVitaleX chloride TB THB chloride in: TB THB ' BA, Blood agar; CA, chocolate agar. The media were incubated at 37 C in candle jars. On agar, the colonies were reported on a basis of 1 + to 4+, by the area in which five or more isolated colonies were observed (1). In broths, the readings were made after their inoculation on BA with % pyridoxal hydrochloride. The five isolates gave similar results. described previously. For this experiment, all of the isolates were freshly retrieved from storage. The need for CO2 was determined with all 22 of the isolates, on blood agar supplemented with 0.002% pyridoxal hydrochloride. Two sets of plates were incubated simultaneously, one in air and the other in candle jars. At the end of the study, all isolates were tested for a possible decrease of their pyridoxal hydrochloride dependence by inoculation on unsupplemented blood agar and chocolate agar. Physiological and biochemical identification. All of the media used for propagation and biochemical characterization of the streptococci were supplemented with 0.002% pyridoxal hydrochloride (5, 19). The tests used in this study were the standard biochemical procedures described previously for the identification of alpha-hemolytic streptococci (10). Hemolysis was read with a dissecting microscope after 24 and 48 h of incubation. Organisms were inoculated on bovine blood agar, and incubation was at 37 C in candle jars. Inocula for differential media were prepared by removal, with a sterile cotton swab, of the growth from an overnight blood agar culture to a tube containing 3 ml of sterile saline (0.15 M). Turbidity was approximately equal to that of an overnight broth culture in THB, i.e., corresponding to about 109 CFU/ml (7). Of this suspension, 0.1 ml was added to each test medium, as well as to a blood agar plate for verification of purity. The differential tests used in this study were as follows: tolerance to 40% bile, methylene blue milk, temperature (45 C), 6.5% NaCl, and 0.1% tetrazolium; hydrolysis of arginine, esculin, hippurate, and starch; production of acid from 10 carbohydrates (see table 2); production of polysaccharide from 5% sucrose; and resistance to bacitracin (0.04 U). Tolerance to bile was determined on bile-esculin agar (Difco) without horse serum (15). Tolerance to 45 C, 6.5% NaCl, and 0.1% tetrazolium (Sigma) was tested in THB. Ability to hydrolyze arginine was tested in Moeller decarboxylase base broth (Difco) with 1.0% L-arginine hydrochloride (Sigma) overlaid with sterile mineral oil. For esculin hydrolysis, heart infusion agar base (Difco) was used with 0.1% esculin and 0.05% ferric citrate. Hydrolysis of hippurate was determined by a rapid method described by Hwang and Ederer (16). Production of acid from carbohydrates was detected in heart infusion broth base with added filtersterilized carbohydrates (final concentration 1.0%) and bromocresol purple as the ph indicator. The 5% sucrose broth used for determination of dextran production and the 5% sucrose agar for the production of levan were prepared as described by Facklam et al. (12). Finally, resistance to bacitracin (0.04 U) was evaluated on blood agar with filter paper discs (Taxo A disks; BBL) (12). Serological grouping. All isolates were tested with the rapid latex test system Streptex (Wellcome Reagents Div., Burroughs Wellcome Co., Research Triangle Park, N.C.) against antisera groups A, B, C, D, F, and G. Antimicrobial agent susceptibility testing. The MICs of penicillin, erythromycin, chloramphenicol, and gentamicin were determined by the agar dilution technique (2). Inocula were prepared from THB cultures supplemented with 0.002% pyridoxal hydrochloride. The turbidity of the 18-h broth cultures was adjusted to match that of a 0.5 MacFarland standard (ca. 108 CFU/ml) (8). The inoculation of agar plates was performed with a Steers inoculum-replicating apparatus delivering ca to ml or ca. 105 viable cells. The spots were applied to Todd-Hewitt agar plates supplemented with 0.002% pyridoxal hydrochloride. The plates were incubated 24 h in air at 37 C. The MIC was defined as the lowest concentration of an antimicrobial agent that prevented macroscopic growth (8). RESULTS Unsupplemented TB and blood and chocolate agars failed to support growth of any of the five isolates. Some slight growth occurred in unsupplemented THB after 48 h (Table 1). On blood agar supplemented with 1.0% IsoVitaleX, growth was slight after 24 h and moderate after 48 h. The addition of 0.002% pyridoxal hydrochloride to the blood agar resulted in moderate growth (3+) within 24 h and heavy growth (4+) after 48 h. In liquid media, growth stimulation by pyridoxal hydrochloride was noticeably more effective in THB than in TB. Results were similar with the five isolates. The growth of all 22 isolates appeared to be stimulated by the presence of pyridoxal hydrochloride. The diameter of the zone of growth varied between 35 and 45 mm, and the growth was more abundant in the peripheral 10 mm of the zone. Of the 22 isolates, the growth of only 2 was adversely affected by the absence of CO2. One did not grow at all, whereas the other grew slightly. At the end of the study, after about 12 subcultures, 21 of the isolates were able to grow on blood agar and on chocolate agar in the absence of pyridoxal hydrochloride. All of the isolates were presumptively identified as viridans streptococci on the bases of gram reaction and morphology (gram-positive cocci in chains), gamma or alpha reaction on blood agar, absence of catalase, and nontolerance to 6.5% NaCl. Moreover, they were all unable to grow in the presence of 0.1% tetrazolium and 40% bile. All failed to hydrolyze hippurate; to produce acid from arabinose, inulin, and melibiose; and to produce polysaccharides from 5% sucrose broth and 5% sucrose agar. On the other hand, they were all positive for the hydrolysis of arginine and esculin, and with a few exceptions they produced acid from lactose, trehalose, and sucrose. Salicin and mannitol gave variable results, as did hydrolysis of starch. Only one isolate grew at 45 C, four grew in methylene blue milk, and two were susceptible to 0.04 U of bacitracin. Finally, one isolate produced acid from both raffinose and sorbitol. The physiological and biochemical patterns of 10 isolates were similar to that of S. intermedius. Nine isolates had identical reactions to the former except that they could produce acid from mannitol. Two isolates were found to be very similar to S. constellatus based mainly on their inability to produce acid from lactose. The raffinose- and sorbitolpositive isolate could not be associated with any of the viridans species. The reactions, along with those of the four reference strains, are shown in Table 2.

3 1132 HIGGINS, BIBERSTEIN, AND JANG J. CLIN. MICROBIOL. TABLE 2. Physiological and biochemical characteristics of 22 NVS isolates from eyes of horses and comparison with four reference strains' Effect Strain SeNo. blood Hydrolysis of: Production of acid from: Biotype N. of Identification isolates agar ax y Arginine Esculin Inulin Lactose Mannitol Raffinose Salicin Sorbitol Sucrose Trehalose Equine A 10 4b S. intermedius Reference S. intermestrain dius SS-898 Equine B Unidentified' Equine C S. constellatus Reference S. constellastrain tus Ss-1111 Equine D Unidentified Reference S. salivarius strain SS-262 Reference S. mitis strain SS-429 a Species of reference strains: SS-898, S. intermedius; SS-1111, S. constellatus; SS-262, S. salivarius; SS-429, S. mitis. For other reactions, see text. b Number of positive, if less than the number of isolates. ' Mannitol-positive S. intermedius. None of the isolates reacted with antisera to groups A, B, C, D, F, and G in the Streptex test. The MICs determined by the agar dilution method are shown in Table 3. Of the isolates, 20 were inhibited by.0.1,ug of penicillin per ml, whereas the remaining 2 and the 4 reference strains were resistant to this concentration but susceptible to 0.5 p.g of this antibiotic per ml. All of the isolates and reference strains had MICs.0.25 plg of erythromycin per ml, the lowest concentration tested. The 22 isolates and the 4 reference strains were inhibited by.4,ug of chloramphenicol per ml. Finally, all of the isolates and three of the reference strains were inhibited by.4,ug of gentamicin per ml, whereas the other reference strain (S. mitis) required a MIC of 8 p,g of this antimicrobial agent per ml. DISCUSSION These appear to be the first reported encounters with NVS in animals. Like their counterparts of human origin (3-5, 9, 14, 17, 19), all 22 strains responded strikingly to pyridoxalhydrochloride supplementation at the recommended rate of 20,ug/ml (0.002%), an amount within the optimal range of 10 to 100,ug/ml reported by Roberts et al. (19). It should be noted that not all forms of vitamin B6 (e.g., pyridoxine hydrochloride; 21) make suitable supplements. Nor did 1% IsoVitaleX elicit growth responses as did pyridoxal hydrochloride. Its effects were probably due to its content of cysteine, a known promoter of NVS growth (5, 19), but IsoVitaleX was required in amounts 100 to 1,000 times those of pyridoxal hydrochloride for equivalent results (19). The use of impregnated disks is suggested as an alternative to the incorporation of pyridoxal hydrochloride into media in some situations. Dependence on increased CO2 tension during primary isolation, as reported for some other viridans streptococci (18, 19), was observed for two isolates. Like the need for pyridoxal supplementation with most strains (9), this dependence disappeared upon repeated subpassages, but both conditions should be met in primary isolation attempts. For some strains, after adaptation to unsupplemented media, altered biochemical test patterns have been reported (9). The recovery of NVS resembling S. intermedius has not, to our knowledge, been reported previously in the medical literature. It accounted for about 45% of our isolates. Nutritionally variant S. constellatus, on the other hand, has been recorded earlier (9). Of our isolates, nine differed from S. intermedius only by their ability to produce acid from mannitol. Ruoff et al. (20) reported the isolation, from urine specimens, of 54 isolates of viridans streptococci resembling S. intermedius but capable of producing acid from mannitol and raffinose. To accommodate these unidentified urine isolates, these authors proposed a third subgroup, the Streptococcus milleri group, in addition to S. intermedius and S. constellatus. Since our nine mannitol-positive isolates did not produce TABLE 3. Effect of four antimicrobial agents on 22 NVS isolates and two strains of viridans streptococci Cumulative % inhibited at Drug Organism MIC (,ug/ml) of: Penicillin NVS Viridansa 90 0 ND" ND Erythromycin NVS ND 100 Viridans ND 100 Chloramphenicol NVS ND ND Viridans ND ND Gentamicin NVS ND Viridans ND a ND, Not determined. b S. salivarius SS-262 and S. mitis SS-429.

4 VOL. 20, 1984 NUTRITIONALLY VARIANT STREPTOCOCCI IN HORSES 1133 acid from raffinose, they appear to be intermediate between this third subgroup and S. intermedius. Nor can they, or our raffinose- and sorbitol-positive isolate, be fitted into the scheme devised by Facklam (10) for the speciation of viridans streptococci. These strains are thus variant not only in their nutritional requirements but also in their biochemical characteristics. The hemolytic reactions of the 22 isolates were about equally divided between alpha-hemolytic and nonhemolytic reactions. This finding is in agreement with previous data concerning S. intermedius and S. constellatus (10). The possibility of group D enterococci was ruled out by the negative results with 6.5% NaCl broth and bile-esculin medium. Two isolates identified as S. intermedius were able to reduce methylene blue milk, a characteristic described by Facklam (10) as typical of 17% of his stock strains of this organismn. Unlike Streptococcus equinus, Streptococcus bovis, and Streptococcus mutans, all of our isolates were bile sensitive (10, 11, 19). Identification with any of these three species as well as with S. morbillorum, S. salivarius, S. mitis, Streptococcus uberis, Streptococcus acidominimus, and S. sanguis II was also ruled out by the positive results with respect to both arginine and esculin hydrolysis, which appear to be important characteristics of our equine NVS. Hydrolysis of starch was variable and of little value in the differentiation of the isolates. This test is known to be a variable characteristic of the viridans streptococci (10). The production of acid from carbohydrates is considered a stable differentiating characteristic (10). In this study, tests on arabinose, inulin, and melibiose gave negative results. Among them, the one on inulin is considered the most important (11) and allowed us to rule out S. salivarius, S. bovis, S. mutans, S. uberis, and S. sanguis I. Reactions in salicin were not very helpful in this study, whereas the 80% of positive results obtained with trehalose separated our isolates from S. mitis, which has been the most frequent isolate of NVS in human medicine (3, 5, 19). Facklam (10) also reported variable results with these two carbohydrates in the S. intermedius group. Production of acid from sucrose, although a fairly constant feature among viridans streptococci, is not an invariable trait of the S. intermedius group, into which our one sucrose-negative strain otherwise fits (10). The absence of reaction in 5% sucrose broth and in 5% sucrose agar further confirmed the nonidentification of our isolates with S. mutans, S. sanguis, S. salivarius, and S. bovis (12). Resistance to bacitracin, used for presumptive differentiation of beta-hemolytic group A from non-group A streptococci (11, 12), is the rule among viridans streptococci, but exceptions occasionally occur (12). Among our isolates, the two susceptible to this antimicrobial agent were among our group of unidentified mannitol-positive NVS. The nonreactivity of our isolates in grouping antisera is in keeping with the recognized lack of group antigens among the viridans species (10). Serology may be helpful to rule out alpha-hemolytic and nonhemolytic strains of groups B, D, N, and Q streptococci from the viridans group (10), including S. equinus and S. bovis. Some authors have, however, reported a few cross-reactions between NVS and antisera against groups H and L (1) and antigens C, E, F, and G (18). The relative resistance of 2 of 22 isolates to penicillin (MIC, >0.1,ug/ml) is in agreement with previous reports (3, 8, 19). Penicillin remains the antibiotic of choice, and a combination with an aminoglycoside has been proposed as an alternative in the treatment of infections due to relatively penicillin-resistant organisms (8). Bouvet et al. (3) noted that NVS and S. mitior (mitis) appeared more resistant to penicillin than did other viridans streptococci. Determinations of MICs for NVS of antibiotics other than penicillin are not well documented. With erythromycin, our results are totally in accord with those of Cooksey and Swenson (8) (MIC, c0.25,.g/ml). The MIC for chloramphenicol (c4 jig/ml) is not significantly lower than that reported by the same authors (c8,ug/ml). A similar value was determined for gentamicin. In conclusion, the NVS of equine origin examined in this study appeared to be similar in their growth requirements to the NVS of human origin described previously (3-5, 7, 9, 13, 14, 17, 19). They showed at least the same degree of physiological heterogeneity, but differed in their remarkable tissue specificity. The pathogenic significance of these NVS from the eyes of horses remains to be studied. Moreover, special alertness to satellitic colonies or the routine use of pyridoxal-hydrochloride supplemented media and CO2 for primary isolation may lead to the isolation of NVS from different specimens and animal species and to a better comprehension of their pathogenicity. The identification of NVS should probably be left to specialized laboratories to which the strains, after a minimum of subculturing, should be referred. ACKNOWLEDGMENTS We are grateful to Eileen Samitz, Laurel Klueh, Anita Wong, Ann Melli, and Bob Miller for their excellent technical assistance in the primary isolation of the streptococci; we also thank Richard R. Facklam for providing the reference strains and for his helpful comments about the manuscript. We acknowledge, with thanks, support through funds from the Equine Research Laboratory, School of Veterinary Medicine, University of California, Davis. LITERATURE CITED 1. Barry, A. L Clinical specimens for microbiologic examination, p In P. D. 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