D. M. MacLaren

Virulence of urinary and faecal Escherichia coli in relation to serotype, haemolysis and haemagglutination

The virulence of faecal and urinary Escherichia coli strains was studied in relation to serotype, haemolysin production and haemagglutination pattern. By means of an experimental mouse model E. coli strains can be divided into avirulent (I), mouse nephropathogenic (II), and generally virulent (III) strains. Virulent group II and group III strains were more often haemolytic and haemagglutinating than avirulent group I strains. Presence of K antigen could not be associated with virulence. Discriminant analysis for qualitative variables revealed that no combination of the investigated properties contributed more to a strains virulence level than did one single property. It is concluded that other virulence factors, apart from haemolysin production in group II strains and haemagglutinins in group III strains, must be involved in the determination of a strains virulence level. All O2, O6 and O18 ac strains tested were virulent, and by far the most O75 strains were avirulent, whereas other O groups were more variable with regard to virulence. Pyelonephritis strains were more often mannose-resistance haemagglutinating than faecal and other urinary isolates, indicating that mannose-resistant adhesins may be important in the pathogenesis of pyelonephritis.

Investigation of the haemolytic activity of Proteus mirabilis strains

Young broth cultures of all P. mirabilis strains tested exhibited haemolytic activity. This activity seemed to be strongly cell-associated as only a very small fraction of this activity was found in the cell-free supernatant. The haemolysin was only produced by actively growing cells. Inhibition studies with trypsin and chloramphenicol suggested that the haemolysin is of protein nature. Lecithin and serum of several species had an inhibitory effect on the haemolysin. Besides erythrocytes of various species also VERO cells were affected by the haemolysin. A correlation was found between the haemolytic activity of a strain and its virulence in an experimental mouse model.

Taxonomy of coagulase-negative staphylococci: a comparison of two widely used classification schemes

In a comparative study 351 strains of Micrococcus subgroups 1–3 (Baird-Parker, 1963, 1965, 1974) were classified according to the Kloos and Schleifer scheme (1975b). The results showed that Micrococcus subgroups 1, 2 and 3 are heterogeneous groups in the Kloos and Schleifer scheme. Strains belonging to Micrococcus subgroups 1 and 2 were mostly classified by the Kloos and Schleifer criteria as Staphylococcus hominis, Micrococcus subgroup 3 strains from the skin as S. cohnii, while Micrococcus subgroup 3 strains from urinary infections were classified mainly as S. saprophyticus. The correlation of novobiocin resistant S. saprophyticus biotype III (Baird-Parker, 1974) with S. saprophyticus (Kloos and Schleifer, 1975a, 1975b) when isolated from urine, was 80%. Although the Kloos and Schleifer scheme provides more information about biochemical characters, doubts are expressed about the validity of some of the species so delineated.

Virulence of Escherichia coli in acute pyelonephritis, acute cystitis and asymptomatic bacteriuria

E. coli strains were isolated from urine specimens of hospitalised patients with acute pyelonephritis, acute cystitis or asymptomatic bacteriuria (ABU), and tested for virulence in an experimental mouse model. Of 12 pyelonephritisstrains 11 were shown to be virulent and 1 avirulent; of 12 cystitis-strains 4 were virulent and 8 avirulent; of 12 ABU-strains 5 were virulent and 7 avirulent. It is concluded that, while no difference in virulence was found between cystitis-and ABU-strains, pyelonephritis-strains were more often virulent than cystitis-and ABU-strains.No associations could be shown between virulence of the isolated strains and the presence of antibody-coated bacteria in the urine. Common urinary O types were not more often virulent than other O types. No relationship was seen between virulence and the presence of K antigen or the presence of particular K types.

Virulence ofEscherichia coli strains isolated from urine of patients with acute cystitis and from faeces of healthy women

E. coli strains were isolated from the urine of patients with acute cystitis in general practice and from the faeces of a comparable reference group of healthy individuals. These strains were serotyped and tested for virulence in an experimental mouse model. Of 30 cystitis-strains 18 were virulent, and of 30 faeces-strains 15 were virulent.It is concluded that the cystitis-strains were not more often virulent than the faeces-strains.O antigens commonly found among urinaryE. coli isolates were present in 60% of the cystitis-strains and in 37% of the faeces-strains. K antigens commonly found in urinaryE. coli strains were present in 33% of the cystitis-strains and in 12% of the faeces-strains. Neither the presence of common urinary O-antigens, nor the presence of common urinary K antigens could be associated with virulence of the isolated strains. However, it is suggested that certain O and K antigens (O2, O6, K23) may be associated with virulence for the urinary tract.

Haemolytic activity of Proteus mirabilis strains

as compared to non-treated bacteria, while adhesion orS. epidermidis (strain 2) grown in TSB with SIC of penicillin onto both FEP and CA was unchanged. Separately, it was found that EDTA did not interfere with the adhesion ofS. epidermidis onto FEP, which indicates that divalent cations are not essential in the adhesion process. Comparison of both S. epidermidis strains and S. saprophyticus in the xylene-affinity test showed that S. saprophyticus has a more hydrophilic cell surface due to the presence of a capsule. Proteinaceous material present at the surface of S. epidermidis is probably digested by pepsin treatment leading to an increased hydrophilicity of the bacterial cell surface. Trypsin or chymotrypsin treatment of the bacteria had no effect. Extraction of LTA and other cell wall components of S. epidermidis by aqueous phenol resulted also in a more hydrophilic bacterial cell surface. Reduced adhesion of bacteria onto FEP is paralleled by an increased cell surface hydrophilicity of the bacteria. The adhesion of bacteria onto polymeric substrates is related to the surface tensions of the bacteria involved and the substrates used. In the present system the adhesion of bacteria onto polymeric films can be described by the thermodynamic approach of Absolom et al. (1979) for the adhesion of cells in a liquid medium to various substrates with various surface tensions. The surface tension of S. epidermidis is less than that of the suspending liquid (PBS), and an increased adhesion onto FEP is predicted in comparison to that onto less hydrophobic CA. The surface tensions of the more hydrophilic S. saprophyticus and pepsin-treated or aqueous phenoltreated S. epidermidis might be close to the surface tension of the suspending liquid, which explains the low adhesion of these bacteria onto both substrates.

Hemolysis and urinary virulence of Escherichia coli.

In a previous study, using a mouse model, we found differences in virulence between different E. coli strains (Van den Bosch et al., 1979). By following the kinetics of the viable count in the mouse kidney and in other organs after intravenous injection, together with LDs0 values and killing rates, we were able to divide E. coli strains into three main virulence groups: Group I strains were avirulent; Group II strains were specifically virulent for the mouse kidney; Group III strains were the most virulent, with a more general virulence for the mouse. Virulent group II and group III strains were more often hemolytic than avirulent group I strains. Hemolytic virulent strains often caused hematuria in the mice, and caused death more rapidly than the non-hemolytic virulent strains. When hemolytic activity was diminished or abolished completely by treatment of the strains with Actinomycin-D, without other properties such as serotype or hemagglutionation pattern being changed, group II strains became avirulent group Itype strains in our mouse model, while the behaviour pattern in the mouse kidney of group III strains did not change. Only the ~leath rate of mice injected with lessor non-hemolytic group III strains decreased compared with the hemolytic wild types, and hematuria was no longer observed when strains became non-hemolytic. We conclude that hemolysin production of E. coli is an important virulence factor in nephropathogenic group II strains, while in the most virulent group III strains it only contributes to the toxicity for mice.

β-Haemolysis by Streptococcus mutans

The pyogenic streptococci from the upper respiratory tract and the oral cavity have attracted considerable interest because of their pathogenic potential. The strains belonging to this group almost invariably are [3-haemolytic, producing haemolysins which have been related to their virulence (Wannamaker and Matsen, 1972). On the other hand, little attention has been paid to the incidence and the nature of haemolysis among other streptococci. [3-Haemolytic strains of Streptococcus mutans can be found in low frequency. We have studied 8 [3-haemolytic strains from the oral cavity. These strains formed acid from mannitol and sorbitol, produced dextrans from sucrose and formed clear haemolytic zones on blood agar plates. The haemolytic zones closely resembled the [3-haemolytic zones produced by the pyogenic streptococci. By using a modification of the method of Ginsburg, Harris and Grossowicz (1963) for the detection of streptolysin S, we were able to solubilize haemolytic activity from the cells of 6 S. mutans strains. The cells were incubated overnight in phosphate buffered saline containing maltose and Tween 80. After titration, haemolytic activity could be demonstrated ranging between 8 and 64 Units/ml depending on the strains tested. One Haemolytic Unit was defined as the amount that completely lysed a red cell suspension (final concentration, 0.5 ~ v/v) within 30 minutes at 37 °C. In contrast with streptolysin S from the pyogenic streptococci, the substitution of other detergents, of human serum or of yeast RNA for Tween 80 (Ginsburg et al., 1963) did not result in solubilization of any haemolytic activity. On the other hand, maltose could be replaced by glucose without a change in haemolytic activity. Furthermore, the haemolytic activity from S. mutans cells was not affected by the addition of Mg ÷ +ions or cysteine. This Tween 80-dependent haemolytic activity remained unchanged after 30 minutes of incubation at 100 °C as well as after 30 minutes of incubation at pH 4 or pH 10. No haemolytic activity could be demonstrated in the supernatants of growing and stationary cultures of the haemolytic S. mutans strains. This observation is in contrast with the results of Wolff and Liljemark (1978), who reported the demonstration of a soluble haemolytic activity in the supernatants of haemolytic S. mutans cultures. Studies are in progress to characterize further this haemolytic activity ofS. mutans and to see whether this property contributes to the virulence of these strains.

Virulence of coagulase-negative staphylococci

In recent years, clinical and laboratory data have been gathered to support the concept that of the S. saprophyticus biotypes it is mainly biotype 3 strains (Baird-Parker, 1974) which are involved in urinary tract infections. These infections are seen for the most part in domiciliary practice in young women. In several studies, S. saprophyticus biotype 3 has been found in 0 to 30 ~ of all urinary tract infections. Although S. saprophyticus biotype 3 is not as frequently found in urinary tract infections as E. coli, it forms a sizeable proportion of such infections and, moreover, the severity of the symptoms is not less. However, the epidemiology of these infections is still unknown and the question arises whether urinary tract infections caused by S. saprophyticus biotype 3 result from its prevalence on the skin of young women or whether specific and more virulent strains are involved. In our preliminary studies we examined the frequency of urinary tract infections by S. saprophyticus biotype 3 in a general practice near Amsterdam. S. saprophyticus biotype 3 strains composed 5 ~ of all the bacteria isolated from urinary tract infections. When strains were arranged according to the sex and age of the patients, this biotype composed 9 % of all the isolates from women of the age of 16-35 years. Our epidemiological studies showed that S. saprophyticus biotype 3 made up only 5 ~ of the bacteria isolated from the skin of males and females and was not the predominant bacterial species. No significant differences were found in numbers of this biotype whether isolated from females or males. L Ds 0 determination of coagulase-negative staphylococci in neonatal mice revealed that strains of S. saprophyticus biotype 3 varied in virulence according to novobiocin sensitivity. Novobiocinresistant strains of S. saprophyticus biotype 3, whether isolated from urinary tract infections or from the skin, showed the same degree of virulence in neonatal mice, whereas novobiocin-sensitive skin isolates were significantly less virulent. Al though extrapolation from animal studies to the human situation is difficult, our LD 50 results suggested some differences in virulence. Therefore, a study of the resistance to phagocytosis and intracellular killing was carried out because these processes are o f vital importance in the body defence system. This might point to biotypes with potentially enhanced virulence. The uptake of strains belonging to a given staphylococcal species or biotype by human polymorphonuclear leucocytes varied from 60 to 80 ~ with an average of 70 % after 20 min incubation at 37 °C. Up to 95 ~ of intracellular bacteria were killed after 20 rain. In spite of the difference in LD s 0 values of novobiocinresistant S. saprophyticus strains compared with other staphylococcal strains (Namavar et al., 1978), there were no marked differences in their uptake and killing by human leucocytes. Why S. saprophyticus biotype 3 should invade the urinary tract of young women is still unknown and further studies might be directed to a study of superficial antigens ofS. saprophyticus biotype 3 in relation to cell adherence.