Identification and Characterization of Sporadic Is
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微生物临床杂志 2005年第2期
Streptococcus Laboratory, Centers for Disease Control and Prevention, Atlanta, Georgia
School of Public Health, University California, Berkeley, California
ABSTRACT
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).
TEXT
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 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 beta-hemolytic 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 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 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. 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 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 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 μg/ml, and those for the other strains were all less than 2.0 μg/ml. The ciprofloxacin MIC for one strain, from a dolphin, was 4.0 μg/ml; those for all other strains were 0.5 μg/ml. There is minimal information in the literature for comparison with 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 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.
REFERENCES
Elder, A., Y. Bejerano, and H. Bercovier. 1994. Streptococcus shiloi and Streptococcus difficile: two new streptococcal species causing a meningoencephalitis in fish. Curr. Microbiol. 28:139-143.
Elder, A., P. F. Frelier, L. Assenta, P. W. Varner, S. Lawhon, and H. Bercouvier. 1995. Streptococcus shiloi, the name for an agent causing septicemic infections in fish, is a junior synonym of Streptococcus iniae. Int. J. Syst. Bacteriol. 45:840-842.
Elliott, J. A., K. D. Farmer, and R. R. Facklam. 1998. Sudden increase in isolation of group B streptococci, serotype V, is not due to emergence of a new pulsed-field gel electrophoresis type. J. Clin. Microbiol. 36:2115-2116.
Facklam, R. 2002. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin. Microbiol. Rev. 15:613-630.
Facklam, R., and J. A. Elliott. 1995. Identification, classification and clinical relevance of catalase-negative, gram-positive cocci, excluding the streptococci and enterococci. Clin. Microbiol. Rev. 8:479-495.
Facklam, R., J. Elliott, N. Pigott, and A. R. Franklin. 1995. Identification of Streptococcus porcinus from human sources. J. Clin. Microbiol. 33:385-388.
Facklam, R. R., T. Thompson, D. Jackson, R. Franklin, and A. Herz. Selection of streptococcal strains to evaluate serologic grouping reagents used to identify group antigens in streptococci, p. 813-817. In D. R. Martin and J. R. Tagg (ed.), Streptococci and streptococcal diseases: entering the new millennium. Securacopy, Auckland, New Zealand.
Fuller, J. D., D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo. 2001. Streptococcus iniae virulence is associated with a distinct genetic profile. Infect. Immun. 69:1994-2000.
Fuller, J. D., A. C. Camus, C. L. Duncan, V. Nizet, D. J. Bast, R. L. Thune, D. E. Low, and J. C. S. de Azavedo. 2002. Identification of a streptolysin S-associated gene cluster and its role in pathogenesis of Streptococcus iniae disease. Infect. Immun. 70:5730-5739.
Goh, S. W., D. Driedger, S. Gillett, D. E. Low, S. M Hemmingsen, M. Amos, D. Chan, M. Lovgren, B. M. Willey, C. Shaw, and J. A. Smith. 1998. Streptococcus iniae, a human and animal pathogen: specific identification by the chaperonin 60 gene identification method. J. Clin. Microibiol. 36:2164-2166.
Johnson, D. R., J. Sramek, E. L. Kaplan, R. Bicova, J. Havlicek, H. Havlickova, J. Motlova, and P. Kriz. 1996. Laboratory diagnosis of group A streptococcal infections. World Health Organization, Geneva, Switzerland.
Lancefield, R. C. 1957. Differentiation of group A streptococci with a common R antigen into three serological types, with a special reference to the bactericidal test. J. Exp. Med. 106:525-544.
Lau, S. K. P., P. C. Y. Woo, H. Tse, K.-W. Leug, S. S. Y. Wong, and K.-Y. Yuen. 2003. Invasive Streptococcus iniae infections outside North America. J. Clin. Microbiol. 41:1004-1009.
National Committee for Clinical Microbiology Standards. 2004. Performance standards for antimicrobial susceptibility testing M2-A8 and M7-A6. 14th informational supplement M100-S14. National Committee for Clinical Microbiology Standards, Wayne, Pa.
Pier, G. B., and S. H. Madin. 1976. Streptococcus iniae, sp. nov., a beta-hemolytic streptococcus isolated from an Amazon freshwater dolphin, Inia geoffrensis. Int. J. Syst. Bacteriol. 26:545-553.
Pier, G. B., S. H. Madin, and S. Al-Nakeeb. 1978. Isolation and characterization of a second isolate of Streptococcus iniae. Int. J. Syst. Bacteriol. 28:311-314.
Thompson, T., and R. Facklam. 1997. Cross-reactions of reagents from streptococcal grouping with Streptococcus porcinus. J. Clin. Microbiol. 35:1885-1886.
Weinstein, M. R., M. Litt, D. A. Kertesz, P. Wyper, D. Rose, M. Coulter, A. McGeer, R. Facklam, C. Ostach, B. M. Willey, A. Borczyk, D. E. Low et al. 1997. Invasive infections due to fish pathogen, Streptococcus iniae. N. Engl. J. Med. 337:589-594.(Richard Facklam, John Ell)
School of Public Health, University California, Berkeley, California
ABSTRACT
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).
TEXT
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 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 beta-hemolytic 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 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 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. 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 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 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 μg/ml, and those for the other strains were all less than 2.0 μg/ml. The ciprofloxacin MIC for one strain, from a dolphin, was 4.0 μg/ml; those for all other strains were 0.5 μg/ml. There is minimal information in the literature for comparison with 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 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.
REFERENCES
Elder, A., Y. Bejerano, and H. Bercovier. 1994. Streptococcus shiloi and Streptococcus difficile: two new streptococcal species causing a meningoencephalitis in fish. Curr. Microbiol. 28:139-143.
Elder, A., P. F. Frelier, L. Assenta, P. W. Varner, S. Lawhon, and H. Bercouvier. 1995. Streptococcus shiloi, the name for an agent causing septicemic infections in fish, is a junior synonym of Streptococcus iniae. Int. J. Syst. Bacteriol. 45:840-842.
Elliott, J. A., K. D. Farmer, and R. R. Facklam. 1998. Sudden increase in isolation of group B streptococci, serotype V, is not due to emergence of a new pulsed-field gel electrophoresis type. J. Clin. Microbiol. 36:2115-2116.
Facklam, R. 2002. What happened to the streptococci: overview of taxonomic and nomenclature changes. Clin. Microbiol. Rev. 15:613-630.
Facklam, R., and J. A. Elliott. 1995. Identification, classification and clinical relevance of catalase-negative, gram-positive cocci, excluding the streptococci and enterococci. Clin. Microbiol. Rev. 8:479-495.
Facklam, R., J. Elliott, N. Pigott, and A. R. Franklin. 1995. Identification of Streptococcus porcinus from human sources. J. Clin. Microbiol. 33:385-388.
Facklam, R. R., T. Thompson, D. Jackson, R. Franklin, and A. Herz. Selection of streptococcal strains to evaluate serologic grouping reagents used to identify group antigens in streptococci, p. 813-817. In D. R. Martin and J. R. Tagg (ed.), Streptococci and streptococcal diseases: entering the new millennium. Securacopy, Auckland, New Zealand.
Fuller, J. D., D. J. Bast, V. Nizet, D. E. Low, and J. C. S. de Azavedo. 2001. Streptococcus iniae virulence is associated with a distinct genetic profile. Infect. Immun. 69:1994-2000.
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