Identification and Characterization of Sporadic Isolates of ...

JOURNAL OF CLINICAL MICROBIOLOGY, Feb. 2005, p. 933–937 0095-1137/05/$08.00⫹0 doi:10.1128/JCM.43.2.933–937.2005 Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 43, No. 2

Identification and Characterization of Sporadic Isolates of Streptococcus iniae Isolated from Humans Richard Facklam,1* John Elliott,1 Lynn Shewmaker,1 and Art Reingold2 Streptococcus Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia,1 and School of Public Health, University California, Berkeley, California2 Received 18 August 2004/Returned for modification 3 October 2004/Accepted 17 October 2004

Seven reference strains and seven clinical isolates of Streptococcus iniae, submitted to the Centers for Disease Control and Prevention Streptococcus Reference Laboratory between 2001 and 2004, were successfully identified by a conventional identification system. The seven randomly submitted clinical isolates were sensitive to ␤-lactams, macrolides, quinolones, and vancomycin. Two of the seven clinical isolates were resistant to tetracycline. All seven strains grew well and multiplied in a phagocytosis assay. One of the seven randomly submitted strains was more similar to the type strain of S. iniae than to the other six strains. The latter six strains were similar if not identical to representative strains from a cluster of disease in Canada (M. R. Weinstein et al., N. Engl. J. Med. 337:589–594, 1997). cluster of cases in Canada (18); and the two original S. iniae isolates from dolphins (15, 16). Table 1 lists the seven randomly submitted cultures of S. iniae used in this study. Six of the seven cultures were from California, and four of those cultures were from the San Francisco area (Active Bacterial Core [ABC] surveillance system). One culture was from Pennsylvania. The first three cultures listed in Table 1 were mistakenly submitted as group A streptococci, and of the last four, two cultures were submitted as beta-hemolytic streptococci unable to identify and two were submitted for confirmation of S. iniae. All of the cultures were isolated from sterile sites: six from blood and one from a cyst. Three patients had sepsis and cellulites: the the others presented with sepsis with pneumonia, cellulites, toxic shock, and an infected mass. The ages of the patients ranged from 60 to 88 years: four were female and three were male. The types of infections and the ages of the patients are very similar to those in other reports (10, 13, 18). The ethnic background of all the patients was Asian. This is also similar to previous reports of infections with S. iniae. One suggestion has been made that people of Asian background may be a risk factor for S. iniae infections because of the method of food preparation and potential of transmission of S. iniae from contaminated fish to humans (13). After the Gram stain and catalase reactions, the hemolytic activity of streptococci on blood agar plates is the most informative test in the identification of unknown cultures. The reason for this is that beta-hemolytic streptococci usually have carbohydrate antigens (group antigens) that can be useful in the final identification of the species. For example, beta-hemolytic cultures with group A antigen are almost always S. pyogenes and beta-hemolytic colonies with group B antigen are almost always Streptococcus agalactiae. Occasionally a betahemolytic culture will not have one of the six common antigens (groups A, B, C, D, F, and G); in this case, further investigation is required to identify the culture. In some reports, S. iniae is reported to be alpha-hemolytic or nonhemolytic, and thus the identification procedure is misguided (18). There are more than 50 non-beta-hemolytic streptococcal species, while there

Little attention was paid to the species Streptococcus iniae until a cluster of cases of invasive disease was associated with this organism in 1995 to 1996 (18). The true incidence of this organism in human infections is still unknown, but it is associated with people who handle fresh fish in the course of food preparation. Most of the documented human infections have been in people of Asian ethnicity (10, 13, 18). The identification of S. iniae is somewhat problematic for the clinical laboratory as this Streptococcus has characteristics similar to those of Streptococcus pyogenes in some respects and has been reported as similar to viridans group streptococci as well (18). In some cases, identifications by automated devices (such as Vitek or Microscan) and rapid phenotypic systems (API 20 Strep or Rapid ID 32) have been erroneous or the organisms have remained unidentified (13, 18). S. iniae was first isolated from performing freshwater Amazon dolphins (15, 16). Later large epizootics in fish were reported (1, 2). No attention was given to a single isolate of S. iniae that was identified by our laboratory in 1991. No other S. iniae cultures were identified by the Centers for Disease Control and Prevention (CDC) laboratory until the cluster of cases in Canada. The identification of the first four isolates by the CDC laboratory was not difficult, because the isolates were beta-hemolytic streptococci that did not have a Lancefield group carbohydrate antigen and were not a member of the Streptococcus anginosus group of bacteria. Beta-hemolytic streptococci without Lancefield group antigens are not common and receive additional testing. The purpose of this communication is to provide a simple identification procedure based on our previous work (4). In addition, we report the antimicrobial susceptibility, clonal aspects, and capacity of S. iniae isolates to grow and multiple in human blood. The following cultures were used in this study: seven randomly submitted cultures between 2001 and 2004; in addition, five cultures (three human and two fish isolates) from the * Corresponding author. Mailing address: Streptococcus Laboratory, Centers for Disease Control & Prevention, Atlanta, GA 30333. Phone: (404) 639-0856. Fax: (404) 639-3123. E-mail: rrf2@cdc.gov. 933

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NOTES TABLE 1. Clinical isolates of S. iniae

State/studya

Culture site

Culture date (mo/day/yr)

Diagnosis

Age (yr)

Sexb

CA/ABC CA/ABC CA/ABC CA/SHD PA/SHD CA/Hosp CA/ABC

Blood culture Blood culture Blood culture Thyroid cyst Blood culture Blood culture Blood culture

8/16/00 5/13/01 6/25/01 5/22/02 8/5/03 11/1/03 1/1/04

Sepsis/pneumonia Sepsis/cellulitis Sepsis/cellulitis Infected mass Toxic shock Cellulitis Cellulitis/sepsis

88 71 69 73 63 60 82

M F F M M F F

a CA, Calif.; PA, Pennsylvania. The CA/ABC cultures were submitted as group A streptococci via the ABC surveillance system. SHD cultures were submitted by state health departments, and the Hosp culture was received directly from a hospital. All seven cultures were isolated from people of Asian ethnicity. All patients were hospitalized, and all survived. b M, male; F, female.

are only 12 species and 6 subspecies of beta-hemolytic streptococci. Hemolysis was determined on Trypticase soy agar plates supplemented with 5% sheep blood (Becton Dickinson, Cockeysville, Md.). Figure 1 shows the hemolytic reactions of typical group A streptococci (S. pyogenes), S. iniae, S. porcinus, S. uberis, and viridans group streptococci. The hemolytic reaction should always be read from the medium surrounding the stab into the agar. The stab into the agar mimics anaerobic incubation, which is the ideal atmosphere for determining the hemolytic reactions of streptococci on blood agar plates. Note that only the S. uberis strain does not have a clear halo around the stab in the blood agar plate. All 14 cultures of S. iniae used in this study were clearly beta-hemolytic in the stabbed area of the blood agar plate. Attempts to demonstrate Lancefield antigens A to G were performed with the CDC capillary precipitin test, using Lancefield hot acid extracts with CDC grouping antiserum. The PathoDx slide agglutination grouping kit (Remel, Inc., Lenexa, Kans.) was also used to demonstrate group antigens. None of 14 cultures used in this study had group antigens when tested with the Lancefield hot acid extraction and using CDC grouping antisera A through G in the capillary precipitin test. Weak group C slide agglutination reactions were observed with six of the S. iniae cultures, including four clinical isolates. One isolate, a culture from the Canadian cluster, reacted weakly with the group F antiserum in the PathoDx test. We interpreted these weak reactions as negative, but an inexperienced microbiologist may interpret them as positive. We have documented cross-reactions with S. porcinus and group B antiserum in several products (6, 17). In fact we have previously identified at least one commercial slide agglutination product that has cross-reactions with group G and S. iniae (7). We would like to reiterate our suggestion that manufacturers of streptococcal grouping reagents incorporate cultures of both S. iniae and S. porcinus in their quality control programs to detect cross-reactions with these two species. Determination of a few phenotypic characteristics will allow for the identification of S. iniae cultures. Conventional tests were performed as previously described (4, 5). Table 2 lists 10 tests that can aid in the identification of S. iniae. In addition to S. iniae, S. porcinus also will not have a demonstrable group antigen, because the antigens (groups E, P, U, and V and new groups 1, 2, and 3) found in this species

FIG. 1. Depiction of the hemolytic reaction of S. iniae, S. pyogenes, S. porcinus, S. uberis, and viridans group streptococci on Trypticase soy agar supplemented with 5% sheep blood. (a) S. pyogenes on the left and S. iniae on the right; (b) S. uberis on the left and S. porcinus on the right; (c) viridans group streptococci on the left and S. iniae on the right.

are not usually included in the battery of antisera used to identify human cultures of beta-hemolytic streptococci. There are three species listed in Table 2 that are beta-hemolytic streptococci and are pyrrolidonylarylamidase (PYR) positive. Since S. iniae strains are PYR positive and half of the S. iniae strains are bacitracin positive, it is understandable how some strains of S. iniae are presumptively identified as S. pyogenes (group A streptococci). S. pyogenes is positive in both these tests nearly 100% of the time. The CAMP test differentiates S.

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TABLE 2. Interpretation and percent positive reactions in conventional tests for identification of pyrrolidonylarylamidase-positive beta-hemolytic streptococci Species (n)a

S. pyogenes (20) S. porcinus (26) S. iniae (14)

Interpretation/% positive byb:

Group carbohydrate antigen

Bac

CM

VP

Hip

Arg

Esc

Str

Sbl

Tre

Rib

A E, P, U, V, new None

⫹/100 ⫺/0 V/50

⫺/0 ⫹/92 ⫹/93

⫺/0 ⫹/85 ⫺/7

⫺/0 V/40 ⫺/0

⫹/95 ⫹/98 v/42

V/20 ⫹/100 ⫹/100

⫺/10 ⫺/0 ⫹/100

⫺/0 ⫹/98 ⫺/0

⫹/100 ⫹/100 ⫹/100

NDc ⫹/98 ⫹/100

a All strains were gram-positive cocci in chains, beta-hemolytic on Frypticase soy–5% sheep blood agar, susceptible to vancomycin in a screen test, failed to produce gas in MRS broth, and yielded positive reactions for PYR and leucine amino peptidase. b Abbreviations: Bac, susceptibility to bacitracin; CM, CAMP reaction; VP, Voges-Proskauer reaction; Hip, hydrolysis of hippurate; Arg, deamination of arginine; Esc, hydrolysis of esculin; Str, hydrolysis of starch; Sbl, Tre, and Rib, production of acid in sorbitol, trehalose, and ribose broth, respectively. c ND, not determined.

iniae and S. porcinus (both positive) from S. pyogenes (negative). The tests for Voges-Proskauer, hydrolysis of starch, and acid from sorbitol differentiate S. iniae and S. porcinus. These conventional tests together with the failure to demonstrate a group reaction have worked very well in the CDC laboratory with more than 60 S. iniae and 40 S. porcinus strains (4; unpublished data). Other investigators have tried various devices to aid in the identification of S. iniae. However, this species is not in the database of any automated devices (Vitek, Microscan, etc.) or

TABLE 3. Phenotypic characteristics of S. iniae determined by reactions in the Rapid ID 32 Strep systema Characteristic

Reaction (%)

␤-Glucouronidase ........................................................................100 Alkaline phosphatase ..................................................................100 Acid from: Ribose .......................................................................................100 Trehalose ..................................................................................100 Sucrose ......................................................................................100 Pullulan .....................................................................................100 Maltose......................................................................................100 Melezitose.................................................................................100 Alanine-phenylalanine-proline arylamidase .............................100 Pyroglutamic acid arylamidase...................................................100 ␤-Glucosidase............................................................................... 93 Acid from mannitol ..................................................................... 93 Gly-tryptophane arylamidase ..................................................... 64 Arginine dihydrolase ................................................................... 50 Acid from glycogen ..................................................................... 50 Methyl-␤-D-glucopyranoside....................................................... 43 ␤-Galactosidase............................................................................ 26 ␣-Galactosidase............................................................................ 14 ␤-Manosidase ............................................................................... 14 ␤-Galactosidase............................................................................ 7 Acid from: Lactose ...................................................................................... 7 Melibiose................................................................................... 7 Sorbitol...................................................................................... 0 Raffinose ................................................................................... 0 L-Arabinose .............................................................................. 0 D-Arabitol ................................................................................. 0 Cyclodextrin.............................................................................. 0 Tagatose.................................................................................... 0 Acetoin production (Voges-Proskaner).................................... 0 Hydrolysis of hippurate............................................................... 0 N-Acetyl-␤-glucosaminidase ....................................................... 0 Urease ........................................................................................... 0 a All identifications were of the “unacceptable” profile as determined by the manufacturer.

rapid identification system (API 20 Strep, Rapid ID 32 STREP, etc). The best possible identification for one of these devices would be “unable to identify or unacceptable profile.” Rapid ID 32 tests (Merieux, Inc., St. Louis Mo.) were performed according to manufacturer’s package insert. We tested all 14 of our isolates in the Rapid ID 32 STREP system. The results we received back from the manufacturer were “unacceptable profile.” It would seem rather simple to incorporate this species into the data bank of the identification systems. The data shown in Table 3 indicate that 20 of the 32 tests are either 100% positive (10 tests) or 100% negative (10 tests). In addition, 5 of the 12 variable tests showed variance in only one test result. A positive identification by one of these devices could be misleading because if the identification is Streptococcus dysgalactiae subsp. Equisimilis, as previously reported (13), the source of the infection would be inaccurate because the latter species is most commonly carried by humans and S. iniae is not. Also, if the identification is S. uberis, as one report indicates (18), this could be misleading because this species has never been documented from a human source. Molecular techniques such as sequencing the 16S rRNA gene (13) or DNA hybridization of the chaperonin 60 gene (10) can be used but are not necessary for most laboratories. Broth dilution antimicrobial susceptibility tests were done according to NCCLS guidelines (14) with customized panels purchased from PML Microbiologicals, Wilsonville, Oreg. The

TABLE 4. Antimicrobial susceptibilies of S. iniae MIC (␮g/ml) Drug

Penicillin Ampicillin Cefuroxime Cefotaxime Cefoxitin Cefazolin Erythromycin Clindamycin Vancomycin Ciprofloxacin Levofloxacin Tetracycline

Range tested

observed

0.008–1.0 0.008–1.0 0.004–1.0 0.004–0.5 0.06–16 0.015–1.0 0.03–32 0.015–32 0.12–2.0 0.12–16 0.25–16 2.0–4.0

ⱕ0.008–0.015 ⱕ0.008–0.015 0.008–0.015 0.015–0.03 0.5–1.0 0.06–0.12 ⱕ0.03–0.12 0.03–0.06 0.25–0.5 ⱕ0.12–4.0a 0.25–0.5 ⱕ2.0–4.0b

a One strain, type strain from dolphin, for which the ciprofloxacin MIC was 4.0 ␮g/ml. The MICs for all other strains were ⱕ0.5 ␮g/ml. b Two strains, both human isolates, for which the tetracycline MIC was 4.0; the MICs for all other strains were ⬍2.0 ␮g/ml.

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TABLE 5. Survival and multiplication of S. iniae in freshly collected human blood Zero time inoculum (CFU) at dilution given

No. of CFU after 3 h

% Survival

Multiplication (no. of generations)

SS1440 (human, Canada) 1:4 1,200 1:16 340 1:64 100

670 320 80

56 94 80

0 0 0

SS1543 (human, Canada) 1:4 1,818 1:16 2,400 1:64 1,200

1,280 2,526 2,608

70 ⬎100 ⬎100

0 0 1

143-01 (human, U.S.) 1:16 TNTCa 1:64 1,960

TNTC 5,760

⬎100b ⬎100

? 2

4780-01 (human, U.S.) 1:16 TNTC 1:64 3,920

TNTC 44,800

⬎100 ⬎100

? 3

4787-01 (human, U.S.) 1:16 TNTC 1:64 2,880

TNTC 12,330

⬎100 ⬎100

? 4

2388-02 (human, U.S.) 1:16 TNTC 1:64 1,568

TNTC 23,520

⬎100 ⬎100

? 4

1056-03 (human, U.S.) 1:16 TNTC 1:64 1,050

TNTC 16,800

⬎100 ⬎100

? 5

105-04 (human, U.S.) 1:16 TNTC 1:16 2,100

TNTC 8,680

⬎100 ⬎100

? 5

4989-04 (human, U.S.) 1:16 2,136 1:64 640

14,880 3,280

⬎100 ⬎100

3 2.5

1120 320

88 41

0 0

SS1056 (dolphin) 1:4 672 1:16 192 1:64 108

48 12 4

7 6 4

0 0 0

SS1123 (dolphin) 1:16 6,000 1:64 2,500

1,400 860

23 34

0 0

2880-96 (fish) 1:16 1,280 1:64 786

a b

TNTC, too numerous to count. ⬎100% survival judged by visual inspection in some cases.

antimicrobial susceptibilities of these 14 strains are unremarkable (Table 4). Using the NCCLS guidelines for streptococci that are not S. pneumonia, these strains are susceptible to ␤-lactams, macrolides, quinolones, and vancomycin. The MICs of tetracycline for two clinical isolates were 4.0 ␮g/ml, and those for the other strains were all less than 2.0 ␮g/ml. The ciprofloxacin MIC for one strain, from a dolphin, was 4.0 ␮g/ml; those for all other strains were ⱕ0.5 ␮g/ml. There is minimal information in the literature for comparison with

FIG. 2. Comparison by PFGE of random clinical isolates to the Canadian cluster of S. iniae. The far-left and far-right lanes contain molecular weight markers. The other lanes are numbered from left to right as follows: lane 1, SmaI digest of representative of Canadian cluster of S. iniae (PFGE pattern 1); lanes 2 to 8, SmaI digests of random clinical isolates 143-01, 4780-01, 4787-01, 2388-02, 105-04, 4989-04, and 156-03, respectively; lane 9, SmaI digest of S. iniae type strain ATCC 29178; lane 10, SmaI digest of S. iniae ATCC 29177; and lane 11, SmaI digest of the Canadian fish isolate (PFGE pattern 2).

these results. Penicillin appears to be the drug of choice for managing S. iniae infections (13). The phagocytosis assay procedure recommended by the World Health Organization (11), which is a method very similar to Lancefield’s original method (12) for testing group A streptococci, was used for demonstrating survival and multiplication in human blood. The results of testing the 12 strains of S. iniae for survival and multiplication in the presence of freshly collected human blood (the phagocytosis assay) are shown in Table 5. We used the classic Lancefield procedure by diluting an exponentially growing culture 105 and then diluting this dilution 1:4, 1:16, and 1:64. These dilutions are made in Todd-Hewitt broth and predictably at least one dilution will give CFU that can be counted in blood agar pour plates. The cultures listed as “human, U.S. ” are the randomly submitted strains from the United States. Each of these seven cultures not only survived but multiplied from 2 to 5 generations in 3 h. The two human isolates from the cluster of cases in Canada (SS1440 and SS1543) survived but did not multiply. Survival was between 56 and 100%. This finding is similar to that reported by the Canadian investigators (8). The two original strains isolated from dolphins (SS1056 and SS1123) did not multiply, and survival was very low: 4 to 34% of the original inoculum. The one representative strain isolated from nondiseased fish in

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Canada (18) survived but did not multiply. Overall these results indicate that the randomly submitted cultures of S. iniae are resistant to phagocytosis and are virulent for humans. We cannot explain why the cultures from the Canadian cluster do not multiple in the phagocytosis assay. We essentially repeated their results (8, 9) showing that their human isolates survive but do not multiply. Genomic DNA isolated from S. iniae strains was prepared for pulsed-field gel electrophoresis (PFGE) based on a previously described procedures (3) with the following modifications. Bacteria were grown for 18 to 24 h at 35°C on Trypticase soy agar plates supplemented with 5% defibrinated sheep blood. The bacteria were removed from the plate with a sterile loop and suspended in 0.5 ml of Tris NaCl buffer (1.0 M NaCl in 10 mM Tris-HCl, pH 7.6). The chromosomal digests were separated by PFGE with a switch time of 0.2 to 25 s for 20 h. PFGE of the randomly submitted strains indicate that six of the seven strains had PFGE patterns identical or nearly identical to those of the strains isolated during the cluster of cases of S. iniae infections in Canada (Fig. 2). The pattern was designated A (8). One isolate, from a case of toxic shock in Pennsylvania, gave a PFGE pattern similar to that of the type strain of S. iniae (lanes 8 and 9, Fig. 2). The same isolate was able to multiply for 5 generations in the phagocytosis assay. It may be that this strain is particularly more invasive for humans. Our finding that S. iniae strains isolated from humans have different PFGE patterns is not unique. Investigators in Hong Kong (13) also reported that their two isolates did not have PFGE pattern A (as reported from Canada). It is difficult to compare PFGE patterns from laboratory to laboratory, but each of the laboratories used the Canadian strains for controls in their experiments. It can be concluded that not all S. iniae isolates from humans are clonal.

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