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Vol. 11. No. 6

Production of Pyrogenic Exotoxins in Group A Streptococci Isolated from Patients in Zagreb, Croatia

an increase in serious infections caused by GAS (8, 9). We report here the toxin profiles of GAS isolated from patients with scarlet fever and invasive streptococcal infections. This is the first report on streptococcal pyrogenic exotoxin production by strains isolated in Croatia.

J. Begovac 1. , B. Gmaj nicki 2, P.M. Schlievert 3, D.R. J o h n s o n 4, E.L. Kaplan 4

Patients and Methods. Over a 13-month period

The pyrogenic exotoxin profiles were determined of group A streptococci isolated from patients in Zagreb, Croatia in the period 1989-1990. A total of 12 strains were studied, five from patients with serious infections and seven from patients with uncomplicated infections. Serotypes M1 and M3 were found in seven (58 %) patients. Seven strains produced exotoxin A and ten strains exotoxin B. The proportion of exotoxin A and B producing strains in patients with severe infections (3 patients respectively) was similar to that found in patients with uncomplicated infections (4 and 7 patients respectively).

In recent years the pattern of infections caused by group A beta-haemolytic streptococci has changed (1, 2). A recrudescence of rheumatic fever has been observed in the USA (1, 2), and there have been reports in many countries of invasive streptococcal infections (1-4). A toxic shock-like syndrome, reminiscent of toxic scarlet fever in the preantibiotic era, has also been described (5, 6). Group A streptococci (GAS) produce three antigenically distinct streptococcal pyrogenic exotoxins (also referred to as scarlet fever or erythrogenic exotoxins), designated types A, B and C. These toxins cause scarlet fever and have been implicated in the toxic shock-like syndrome. Studies conducted in the USA have shown a prevalence of strains producing exotoxin A in patients with severe streptococcal infection (6), whereas studies in the UK have shown a predominance of strains producing exotoxin B in similar cases (7). At the University Hospital of Infectious Diseases in Zagreb, we have also noted 1University Hospital of Infectious Diseases "Dr Fran Mihaljovic", Mirogojska 8, 4100 Zagreb, Croatia. Institute of Immunology,Roekefellcrova2, 41000Zagrcb, Croatia. 3Department of Microbiology, and 4Department of Pediatrics and WHO CollaboratingCenter for Reference and Research on Streptococci,University of Minnesota Medical School, Minneapolis, Minnesota, USA.

(June 1989-June 1990) strains of GAS isolated from patients with various streptococcal infections were collected at the Department of Microbiology, University Hospital of Infectious Diseases, Zagreb. Five strains were from patients with serious GAS infections and seven strains from patients with uncomplicated infections (6 with mild scarlet fever and 1 with erysipelas). Thus a total of 12 strains were studied. Clinical data was obtained by reviewing the charts of the patients. The strains were isolated from cultures of blood (n = 1), cerebrospinal fluid (n = 1), joint fluid (n = 1), skin lesion material (n = 1) and throat specimens (n = 8). Organisms suspected of being streptococci were identified according to standard bacteriologic criteria. After serological identification of the GAS using a commercial test-kit (Slidex Streptokit, bioMfrieux, France), all strains were lyophilized. The GAS strains were then forwarded to the WHO Collaborating Center for Reference and Research on Streptococci, University of Minnesota, Minneapolis, MN, USA where strains were typed by M protein, T-agglutination pattern and opacity factor. The toxin studies were carried out in the Department of Microbiology, University of Minnesota, Minneapolis, MN, USA. The production of exotoxin A, B and C was detected by double immunodiffusion as described previously (6). In addition, strains were tested for the presence of speA and speC genes coding for exotoxins A and B respectively using the Southern blot technique and genetic probes (10). The speA probe consisted of approximately 500 base pairs within the speA gene (11). The speC probe was prepared as follows: pUMN 521 (12) was digested with the restriction enzyme DdeI to obtain a 654 base pair fragment which included a large part of the speC structural gene and 34 base pairs 3' to the speC stop codon. All GAS tested so far contain a single copy of the speB gene, and the strains examined in this study were therefore not probed for speB.

Results and Discussion. All cases of streptococcal infection were community acquired and isolated. Clinical and microbiological data are summarised in Table 1. Five patients had a serious

• Vol. 11, 1992

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Table 1: Characteristics of isolates and clinical data of 12 patients with group A streptococcal infection. Patients no. 1 to 5 were considered to have serious infections.

Patient no. 1 2 3 4 5 6 7 8 9 10 11 12

Age (years) Clinical presentation and sex of infection 27/M 66/F 32/M 3/M 3IF 25/F 20/F 8/M 3/M 6/M 8/M 3/F

cellulitis (leg) meningitis purulent arthritis (knee) toxic scarlet fever toxic scarlet fever mild scarlet fever erysipelas (leg) mild scarlet fever mild scarlet fever mild scarlet fever mild scarlet fever mild scarlet fever

Source of isolate

Type

blood cerebrospinal fluid joint fluid throat throat throat

skin throat throat throat throat throat

Exotoxin production

M

T

OF

A

B

C

1 ND 1 ND 3 3 ND ND 3 3 ND 1

1 NT 1 4 3 3/B 12 4 3/B 3 28 1

NA NT NA 4 NA NA 76 4 NA NA 28 NA

+ + NG + + + + +

+ + + NG + + + + + + +

NG _ + -

ND = not determined; NT = not typable; NA = not available; NG = no growth.

G A S infection (patients no. 1 to 5); one died (no. 2). T h e site of infection in these patients was the skin in two cases and the throat in two cases; in one patient (no. 2) the site could not be determined. O f these five patients with serious G A S infections two had predisposing factors (liver cirrhosis in o n e and chronic alcoholism in the other). All strains were bacitracin-sensitive, Lancefield g r o u p A streptococci. Seven of the 12 strains were type M1 or M3 (Table 1). All of the type M1 and M3 p r o d u c e d exotoxin A; six of t h e m also p r o d u c e d exotoxin B. Ten of the 12 strains p r o d u c e d exotoxin B. Only one strain was shown to carry the speC gene. Five strains p r o d u c e d one type of exotoxin and five strains two types of exotoxin. O n e strain, in addition to producing exotoxins A and B, also carried the speC gene. Because of lack of growth in the m e d i u m used, one strain could not be examined for toxic production. T h e toxic profiles in strains f r o m patients with serious and uncomplicated G A S infections are p r e s e n t e d in Table 2. Five patients had serious G A S infections, three of w h o m w e r e hypotensive. H o w e v e r , several imp o r t a n t features characteristic of the toxic shocklike s y n d r o m e (acute renal failure, respiratory distress and disseminated intravascular coagulation) (6) w e r e not observed. This might be due to early institution of t r e a t m e n t with fluids and antibiotics: three of the five patients received treatm e n t within the first 12 hours of their illness. Although the toxic shock-like s y n d r o m e is usually p r e c e d e d by soft tissue infection, the throat might also be the origin of severe G A S infection (6). T h e experience of ourselves and others suggests

that the respiratory tract might be a frequent source of severe G A S infection in children (4, 8). It is k n o w n that the M protein is a m a j o r virulence antigen of G A S . Virulence is believed to be associated with the antiphagocytic activity of the M protein and with the a m o u n t of hyaluronic acid in the capsule (K. Belani et al., Pediatric Research 1988, 23:364A, Abstract no. 978) and p r o b a b l y also with exotoxin production. R e c e n t studies of strains f r o m patients with severe infections show a p r e d o m i n a n c e of types M1 and M3 in the U S A (1; D.R. Johnson et al., l l t h Lancefield International S y m p o s i u m on Streptococci and Streptococcal Disease, 1990, Abstract no. P5), and of type M1 in the U K (7) and Scandinavia (4). The majority of the strains in this study belonged to serotypes M1 and M3. However, because of the small n u m b e r of strains studied no definitive conclusion a b o u t the prevalence o f certain serotypes in Z a g r e b can be made. A change in the patterns of production of streptococcal pyrogenic exotoxins has also been suggested. In the beginning and middle of this century exotoxin A production was prevalent in clinical isolates in the U S A and E u r o p e . However, studies of 80 strains collected b e t w e e n 1976 and 1986 in the U S A (5) and of 40 strains collected between 1980 and 1985 in the U K (13) were not able to d e m o n s t r a t e exotoxin A production. Recently, r e a p p e a r a n c e of strains producing exotoxin A was o b s e r v e d in connection with the shock-like s y n d r o m e in the U S A (6). This contrasts with m o r e recent observations in the U K where none of 32 strains of G A S isolated f r o m fatal infections produced exotoxin A, and 23 p r o d u c e d exotoxin

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Eur. J. Clin. Microbiol. Infect. Dis.

Table 2: Exotoxin profiles of strains from patients with serious and uncomplicated group A streptococcal infection. Exotoxin

Total no. of patients

Aa

Bb

Cc

Serious infection

3

3

0

4

Uncomplicated infection

4

7

1

7

Total

7

10

1

11

aDetected by genetic probes and toxin tests. bDeteeted by toxin tests only. e Detected by genetic probes only.

B (7). Although 10 o u t of 11 of our strains produced exotoxin B, the prevalence of exotoxin A production in our strains is also striking (7 of 11 strains). Interestingly, all our strains of serotype M1 and M3 produced exotoxin A. Similar findings have been observed in the U S A (6). A strong association of the speA gene with serotypes M1 and M3 has also been reported (14). However, since the introduction of the gene probe technique it has been established that many strains demonstrated to have the speA gene produce little or no toxin detectable by standard serological tests (14). This finding suggests that the genetic, bacteriophage-mediated ability of GAS to produce exotoxin A might not have changed over the last century. It is not clear why some patients with strains producing exotoxin A have mild scarlet fever whereas others suffer a lifethreatening illness. Since some strains of G A S in the preantibiotic era produced large amounts of exotoxin A it has been suggested that the severity of the illness is related to the quantity rather than quality o f exotoxin (3). However, in recent reports of cases of toxic shock-like syndrome no relationship between the quantity o f exotoxin produced and the severity of disease could be established (15). In our small study 3 of 4 patients with severe infection had GAS producing exotoxin A whereas 4 of 7 patients with milder infections had strains producing exotoxin A. Similarly, no difference was observed between serious and uncomplicated illnesses in exotoxin B production. Therefore, in our study no definitive conclusion about an association between toxin type and severity of G A S infections can b e made. Larger studies including patients with mild infections and uncomplicated pharyngitis are n e e d e d to determine the relevance of pyrogenic exotoxins for the severity of G A S infection.

References 1. Schwartz B, Facklam RR, Breiman RF: Changing

2.

3. 4. 5.

epidemiology of group. A streptococcal infection in the USA. Lancet 1990, 336: 1167-1171. Kaplan EL: The resurgence of group A streptococcal infections and their sequelae. European Journal of Clinical Microbiology and Infectious Diseases 1991. 10: 55-57. Krhler W: Streptococcal toxic shock syndrome. Zentralblatt ftir Bakteriologie 1990, 272: 257-264. Martin PR, Hoiby EA: Streptococcal A epidemic in Norway 1987--1988.Scandinavian Journal of Infectious Diseases 1990, 22: 421--429. Cone LA, Woodard DR, Schlievert PM, Tomory GS: Clinical and bacteriological observations of a toxicshock like syndrome due to Streptococcus pyogenes. New England Journal of Medicine 1987, 317: 146-149.

6. Stevens DL, Tanner MH, Winship J, Swarts R, Rles

KM, Schlievert PM, Kaplan E: Severe group A streptococcal infections associated with a toxic shock-like syndrome and scarlet fever toxin A. New England Journal of Medicine 1989, 321: 1-7. 7. Gaworzewska ET, Hallas G: Group A streptococcal infections and a toxic shock-like syndrome. New England Journal of Medicine 1989, 321: 1546. 8. Begovac J, Gmajni~ki B, Kuzmanovi~ N: Invasive streptococci. Lancet 1990, 335: 109, 9. Begovae J, Marton E, Lisi~ M, Beus I, Bo~inovi~ D,

Kuzmanovi~ N: Group A beta-hemolytic streptococcal toxic shock-like syndrome. Pediatric Infectious Disease Journal 1990, 9: 369-370. 10. Southern EM: Detection of specific sequences among DNA fragments separated by gel eleetrophoresis. Journal of Molecular Biology 1975, 98: 503-517. 11. Johnson LP, Tomai MA, Schllevert PM: Bacteriophage involvement in group A streptococcal pyrogenic exotoxin A production. Journal of Bacteriology 1986, 166: 623-627. 12. Goshorn SG, Bohach GA, Schlievert PM: Cloning and eharacterisation of the gene, speC for pyrogenic exotoxin type C from Streptococcus pyogenes. Molecular and General Genetics 1988, 212: 66-70. 13. Hallas G: The production of pyrogenic exotoxins by group A streptococci. Journal of Hygiene 1985, 95: 47-57.

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14. Yu CE, Ferretti JJ: Frequency of the erythrogenic toxin B and C genes (speB and speC) among clinical isolates of group A streptococci. Infection and Immunity 1991, 59: 211-215. 15. Chomarat M, Chapius C, Lepape A, Bernard F: "I~vo cases of severe infection with beta-haemolytic group A streptococci associated with a toxic-shock-like syndrome. European Journal of Clinical Microbiology and Infectious Diseases 1990, 9: 901-903.

In Vitro Antimicrobial Susceptibility of Viridans Streptococci Isolated from Blood Cultures E. Potgieter*, M. Carmichael, H.J. Koornhof, L.J. Chalkley

The susceptibility of 211 viridans streptococci isolated from blood cultures to eight antimicrobial agents was determined. All the isolates were susceptible to cefotaxime, ceftriaxone, imipenem and vancomycin. Thirty eight percent of the iso. lares were resistant to penicillin (MICs ___0.25 lag/ml). Tetracycline resistance was found in 41% of the isolates and in 7 % of these strains tetracycline resistance was combined with erythromycin resistance. Five Streptococcus mitis isolates exhibited increased (MIC 64 lag/ml and 128 lag/ml) or high-level (MIC _>500 lag/ml) resistance to gentamicin, kanamycin and tobramycin. Four of these isolates were also resistant to penicillin (MICs 16-32 lag/m). In vitro synergy was not demonstrated for combinations of penicillin and gentamicin against three Streptococcus mitis isolates with gentamicin MICs of 1000, 128 and 64 lag/mi. Results of this study indicate the importance of monitoring antibiotic resistance trends in viridans streptococci particularly with respect to penicillin and aminoglycoside resistance.

Antibiotic resistance in viridans streptococci isolated from clinically significant infections has been reported to be increasing in America and Spain (1; E. Escribano et al., 13th Interscience Emergent Pathogen Research Unit of the South African Medical Research Council, School of Pathology of the University of the Witwatersrand and the South African Institute for Medical Research, 7 York Road, Parktown, Johannesburg 2193, South Africa.

543

Conference on Antimicrobial Agents and Chemotherapy, Atlanta, 1990, Abstract no. 695). In 1978 ten nasopharyngeal viridans streptococci isolated in South Africa were found to be multiply resistant to beta-lactam antibiotics (2). A survey conducted in 1987 of antibiotic resistance in oropharyngeal viridans streptococci isolated from children admitted to Baragwanath Hospital showed that 76 % of the isolates were resistant to penicillin (MICs > 0.25 lag/ml) (3). In this group of isolates a high-level gentamicin-resistant (MIC > 2560 lag/ml) viridans streptococcus was also identified. The possibility of penicillin resistance being coupled to gentamicin resistance in oral strains of viridans streptococci is cause for concern. Such strains, which could give rise to serious infections, would no longer respond to penicillin/ gentamicin combination therapy. This has been found with enterococcal endocarditis (4). The present study was undertaken to investigate the antimicrobial susceptibility of 211 viridans streptococci isolated in South Africa from blood cultures. Materials and Methods, Two hundred and eleven viridans streptococci isolated from blood cultures were collected from three Johannesburg hospital laboratories between June 1988 and March 1991. Strains were identified using conventional biochemical tests (5) and were generally classified according to the criteria of Facklam and Washington (6). Aesculin-positive and argininenegative strains were included in the Streptococcus mitis group and aesculin-negative and arginine-positive strains incorporated into the Streptococcus sanguis group as described by Price et al. (7). The MICs were determined by the agar dilution method (8) using Mueller-Hinton agar (Oxoid, UK) supplemented with 5 % horse blood. Plates were incubated aerobically at 35 °C for 18 h. The antimicrobial agents were obtained as standard reference powders from the respective manufacturers. MICs of penicillin, imipenem, cefotaxime, ceftriaxone, vancomycin, erythromycin, tetracycline and gentamicin were determined using antibiotic concentrations ranging from 0.03 to 128 lag/ml. Strains with gentamicin MICs of >_.64 /ag/ml were further tested at concentrations of 250, 500, 1000 and 2000 lag/ml. MICs of the other aminoglycosides (streptomycin, kanamycin, amikacin and tobramycin) were determined at concentrations of 1000 and 2000 /ag/ml. One strain for which the streptomycin MIC was > 2000 lag/ml was tested for ribosome associated resis-