Cell Surface Hydrophobicity of Group D and Viridans Streptococci Isolated from Patients with Septicaemia

Zbl. Bakt. Hyg. A 261, 280-286 (1986)

Cell Surface Hydrophobicity of Group D and Viridans Streptococci Isolated from Patients with Septicaemia ASA LJUNGHl, MONICA OSTERLIND 1, and TORKEL WADSTROM 1 1

1

Stockholm County Council Central Microbiological Laboratory, Stockholm Department of Veterinary Microbiology, Biomedicum, Uppsala, Sweden

Received October 10, 1985 . Accepted December 4, 1985

Abstract Sixty-three strains of Group D streptococci and viridans streptococci isolated from blood cultures during a two year period were typed to the species level with conventional biochemical tests and API Strep. Streptococcus faecalis was the most common species isolated followed by S. sanguis, S. mitis and S. constellatus (S. milleri). One of the two isolates of S. faecium was a contamination. The reported increasing frequency of this organism and other Group D and viridans streptococci as well as the association of S. bovis with malignant bowel disease indicate the need for full identification of streptococcal isolates from blood cultures. Pronounced surface hydrophobicity as measured with the salt aggregation test (SAT) was expressed by 59/63 (94%) of the blood culture isolates whereas strains isolated from commercial fermentation products and strains passaged several times were hydrophilic. In the presence of human serum albumin which binds to lipoteichoic acid only one strain decreased in surface hydrophobicity. The surface hydrophobicity of two strains even slightly increased indicating that lipoteichoic acid but marginally contributes to surface hydrophobicity of streptoccal cells from these species.

Zusammenfassung Ober einen Zeitraum von 2 Jahren wurden 63 Stamrne von Streptokokken der Gruppe D und Viridans-Streptokokken aus Blutkulturen isoliert und mittels konventioneller biochemischer Tests und API-20-Strep-Test identifiziert. 1m Vordergrund stand Streptococcus faecalis, gefolgt von S. sanguis, S. mitis und S. constellatus (S. milleri). Mit dem SalzAggregations-Test (SAT) wurde bei 53/63 (94%) Stammen eine ausgepragte OberflachenHydrophobizitat der aus dem Blut isolierten Starnrne nachgewiesen, wogegen Starnrne aus Silage oder Nahrungsmitteln sowie mehrfach auf Nahrboden passagierte Keime sich als hydrophil erwiesen. SAT's in Gegenwart von humanem Serumalbumin legen den Schluf nahe, daB Lipoteichonsaure kaum zur Oberflachen-Hydrophobie dieser Streptokokkenzellen beitragt.

Cell Surface Hydrophobicity of Group D and Viridans Streptococci

281

Introduction Studies more than two decades ago showed that enterococci comprise 5-15 % of all cases of endocarditis (10, 16). Other studies have shown that Lancefield group D streptococci (Streptococcus faecalis, S. [aecium, S. bouis, S. durans, S. avium and S. equinus) are common isolates from blood cultures (17, 18, 19) but Kraus e stated that enterococcal infections are usually benign (12). Bloodborne and urinary tract enterococcal infections are often opportunistic infections (6). Granato and Ellner (7) showed a correlation between the isolation of certain species of group 0 streptococci and clinical disease. S. faecalis, S. faecium and S. bov is were the three most common species of group 0 streptococci isolated. Also non-group 0 streptococci (Lancefield groups L to T ) may cau se endocarditis in debilitated patients (2). Although in man y stu dies the majority of viridan s and group 0 streptococci are regarded as contaminants upon isolation from blood cultures (6, 20 ), recent reports have reevalu ated the clinical significance of these species as opportunistic pathogens. S. faecium has been reported as the causative agent of no socomial bacteraemia and meningitis in a neonatal intensive care unit (4 ) while S. mitis was the single most frequent species of viridans streptococci isolated from neonates in one study (9). Very little is known about the virulence factors of group 0 streptococci and viridans streptococci. In a recent st udy o f Staphylococcus aureus isolated from septicaemia, wound and urinary tra ct infections, the ma jor ity of str ains (- 90% ) were found to express high surface hydrophobicity as measured by the Salt Aggregation Test (SAT) whereas cells of staphylococcal strains isolated from the nose of health y car riers were more hydrophilic when grown under similar condition s (13). In this study, group 0 and virid ans st repto cocci isolated from blood cultures du ring a two yea r per iod were typed to the species level and their cell surfac e properties were studied with the SAT, previously shown to be a rapid, simple test to determine relative bacterial cell surface hydrophobicity (13, 14).

Materials and Methods Bacterial strains. Sixty-three strains were isolated from 63 blood cultures from patient s (0-91 years old, mean 39 years of age) in paediatr ic and adult wards in Stockholm hospitals over a two year period. Thirry strains were isolated from the urine of patients with urinary tract infections (?; 105 bacteria/ml). The strains were initially tested in the rout ine laboratory for esculin hydrolysis, acid production from raffinose, and the antibiotic sensitivity pattern was determined. In this study strains were subjected to biochemical testing according to ASM Manual (5). All strain s were also tested with API Strep (API System S. A., La Balme-Ies Grottes, France) according to the manufacturor's instruction . Three S. faecium strains were a kind gift from Dr S. Tabaqchali, UK, and 8 S. faecium strain s were previously isolated from patients with urinary tract infection. In addition, 9 strains of S. faecium were isolated from commercially available products for ensilage or fermentat ion. Strains were stored in Trypticase soy broth (TSB, Oxoid Ltd, London ) with 20% glycerol at - 70 "C. Growth conditions. Strains were grown on blood agar (Oxoi d) with 5% horse erythrocytes at 37 °C for 18 h. Six strains were also grown in 20 ml Brain heart infusion broth (BHI, Difeo, Detroit, Mich.) in 200 ml flasks at 37 °C for 16 h under slow shaking. Salt aggregation test (SAT). Bacterial cells were suspended in 0.001 M sodium phosphate buffer (pH 7.0), washed in this buffer and diluted to 108 bacteria/ml (13, 14). From broth cultures ten ml samples were centrifuged (2.000 x g, 10 min., 4 °q, washed in phosphate

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buffer and diluted to 10 8 bacteria/m!. From a series of ammonium sulphate of different molarities (0.02-4.0, pH 6.8) four concentrations, 0.02, 0.2, 1.8 and 3.2 M were chosen as representing breakpoints for groups with differing hydrophobicity (14). Bacterial suspensions, 10 ul, were mixed with equal volumes of ammonium sulphate solutions of varying molarity on a glass slide and observed for aggregation after 1 min at room temperature. The highest dilution of ammonium sulphate (final concentration) which gave visible aggregation was scored as a numerical value for bacterial cell surface hydrophobicity, the SAT value. Sixteen agar-grown strains were tested in the presence of albumin (1 mg/ml and 5 mg/ml final concentration), and six of these also after growth in broth. Chemicals. Ammonium sulphate, esculin and raffinose, all of analytical grade were purchased from Merck AG, Darmstadt, FRG. Human serum albumin was from KABI, Stockholm, Sweden.

Results The distribution of species isolated in blood cultures typed according to ASM Manual and API Strep is presented in Table 1. Based on colony morphology, esculin hydrolysis, acid production from raffinose and antibiotic sensitivity pattern, in the clinical routine laboratory one S.bovis had been identified as group D streptococcus, and one S. bovis as "alpha-streptococcus". One S. faecalis was misidentified as "alpha-streptococcus" and 2 as S. bovis. Children with heart disease grew S. constellatus (see above) (4 patients), S. sanguis II (2 patients), and S. mitis (2 patients). Of these, one child who underwent cardiac surgery grew S. mitis in the first set of blood cultures, and one week later S. sanguis. Patients with diagnosed malignancies grew S. faecalis (2 patients) and S. bovis (2 patients). Table 1. Streptococcal species isolated from blood cultures

S. bovis S. constellatus (S. milleri) S. equinus S. [aecalis S. faecium S. mitis

S. salivarius S. sanguis I and II Streptococcus Group L

2 5

1

26 2 6 2

18 1

(1) (1) (1) (1)

The numbers in parenthesis denote a probable contamination

One isolate each of the following species was regarded as contaminant: S. faecalis, S. faecium, S. constellatus, and S. salivarius. Insufficient clinical data on another 14 samples cannot rule out that they are also contaminants. S. faecalis and a gramnegative coliform rod were repeatedly isolated concomitantly in five blood cultures in one patient. S.sanguis was isolated together with Staphylococcus epidermidis three times in patients with osteomyelitis.

Cell Surface Hydrophobic ity of Group D and Viridans Streptococci

283

The group D and viridans streptococci isolated from patients with septicaemia or urinary tract infection generally expre ssed pronoun ced surface hydrophob icity as measured by SAT (T able 2). Of the 63 strains isolated from blood cultures in our laboratory, 59 (94%) strains were po sitive in SAT, i.e. aggregated in amm onium salt solutions with molarity of 3.2 or lower (T able 2). O f these, 5 1 strains (81 % ) were autoaggregating, i.e. aggregated in saline , 0.2 and 0.02 M ammonium sulpha te. Tw enty-eight of 30 strains isolat ed from urine were positive in SAT, and all but one of these were autoaggregating, In contrast, 9 str ains of S. faecium isolated from commercial products for ferment ation and ensilage expressed a hydrophilic cell surfa ce and wer e negati ve in SAT. Three strains of S. faecium aggregated in 3.2 M and 0.0 2 M ammonium sulphate respecti vely. The number of times these strains had been subcultured after isolation, and conditions for storage are not known.

Table 2. Surface hydrophobicity of streptococci as measured by the Salt Aggregation Test (SAT) Species

~

S. faecalis (blood) S. [aecalis (urine) S. sanguis S. [aecium S. mitis S. constellatus (S. milleri) S. bovis S. salivarius S. equinus Streptococcus Group L

16 23 18 1b 6 3 2 2 1 1

a b

0.01 M

0.1 M

0.9 M

1.6 M

> 1.6 M

1 4

4

2 1

3 2

2b

9'

1

Isolated from commercial produ cts for fermentation and ensilage. Strains obtained from Dr S. Tabaqchali, London.

Cell sur face hydrophobicity decr eased after repeated subculture on both blood agar and in BHI broth. There was a gradual decrea se in hydrophobicity, e.g, 2 strains wh ich initially aggregated in 0.02 M ammonium sulphate aggregated in 1.8 M solution after 9 subcultures on blood agar, and in 3.2 M after another 8 sub cultures. After 4 further subcultures they were negative in SAT. Several strains decre ased more rapidly in surface hydrophobicity after sub culturing in broth than on agar (dat a not shown). To estimate the influence of cell surface exposed lipoteichoic acid (LTA) on the surf ace hydrophobicity, SAT wa s perf ormed in the presence of hum an serum albumin at 1 and 5 mg/ml final concentration. Six stra ins grown on blood agar did not differ in sur face hydrophobicity when measured with and without albumin (Ta ble 3). After gro wth in BHI broth 2 strains were more hydrophobic in the presence of albumin, and one S. faecalis was more hydrophilic. Three stra ins did not change their surface hydrophobicity during the test procedures.

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and T. Wadstrom

Table 3. Influence of albumin on streptococcal surface hydrophobicity as determined with the Salt Aggregation Test (SAT) Strain

Agar grown bacteria SAT SAT w. albumin"

S. sanguis 1674 S. [aecalis 2167 S. [aecalis 2487 S. [aecalis 161 S. faecalis 1690 S. sanguis 3970

:s;; :s;; :s;; :s;; :s;;

a

0.Q1

0.01 0.01 0.01 0.01 0.01

:s;; :s;; :s;; :s;; :s;;

0.01 0.01 0.01 0.Q1

0.01 0.01

Broth grown bacteria SAT SAT w. albumin" :s;; 0.Q1

> 1.6

:s;; 0.01

> 1.6

1.6 0.01

:s;; 0.01 1.6 1.6 > 1.6 1.6 :s;; 0.01

SAT performed in the presence of human serum albumin, 5 mg/ml final concentration.

Discussion This study confirms previous reports that S. faecalis is by far the most common group D streptococcus isolated from blood cultures of patients with septicaemia (10, 16-19). However, in one American study S. faecium was the second most common species isolated, accounting for 5-15% of the isolates (19). Of the two isolates of S. faecium in this study one is probably a contaminant. The spectrum of pathogens may shift over the years as shown by recent reports on increasing frequency of neonatal septicaemia caused by non -group D alpha-haemolytic streptococci like S. mitis (9), and on a nosocomial outbreak of septicaemia caused by S. faecium (4). These reports as well as the association of S. bovis with bowel disease (11) emphasize the importance of full identification of streptococcal isolates from blood cultures. We find API Strep to be a valuable tool in this routine speciation. These streptococcal species are frequently contaminants in clinical samples. Repeated isolation of streptococci with the same API profile may aid the interpretation of the significance of the isolate. Few studies have been confined to virulence factors of these streptococcal groups. The expression of a hydrophobic bacterial cell surface was earlier suggested as an advantage in the colonization of the intestinal and urinary tracts (14). It may also enhance colonization of intravascular catheters (15). In the present study the majority of strains isolated from infections were hydrophobic whereas strains isolated from commercial products such as S. [aecium M74 (23) were less hydrophobic as determined by SAT. The latter strains had been subcultured many times in contrast to the isolates from patients which probably causes a decrease in cell surface hydrophobicity (24). Lipoteichoic acid (LTA) on the group A streptococcal cell surface binds albumin and has been suggested to be a major determinant of surface hydrophobicity (1). Our finding that the surface hydrophobicity of the two group D strains increased in the presence of albumin, and decreased only slightly in one strain (Table 3) indicates that LTA does not contribute significantly to cell surface hydrophobicity. Streptococci of groups A, C, and G but not of groups Band D were shown to bind fibronectin to their surface (21). It remains to be investigated if the streptococcal strains in the present study bind to other connective tissue proteins which may be exposed, e.g. vascular lesions in heart disease. Careful case histories in two reports suggest that the portal of entry of S. faecalis and S. [aecium into the blood stream is the gastrointestinal tract,

Cell Surface Hydrophobicity of Group D and Viridans Streptococci

285

and that patients with underlying gastrointestinal disease are at risk. These observations seem quite important in view of present attempts to prove the efficacy of S. faecium strains originally isolated from human intestinal tracts as prophylaxis against travellers' diarrhoea and neonatal diarrhoea in piglets (3, 22). The high resistance of irradicated mice (sublethal dose) to iv injection of S. faecium strain M74, and the lack of side effects in young piglets (22) and young rabbits treated (23) suggest that S. faecium is non-virulent in normal, non-debilitated animals. The strains are probably attenuated by multiple laboratory passages in the preparation of commercial, lyophilized tablets. However, until strains of S. faecium to be used prophylactically have been tested in other animal models like the rabbit endocarditis model (8) we believe, that patients with underlying disease or premature infants should restrain from using this preparation which is now regularly recommended in several countries.

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