Unusual antineisserial activity expressed by a systemic isolate of Neisseria meningitidis. antimeningococcal effect and properties.

Antineisserial activity expressed by the systemic Neisseria meningitidis strain 77/79A was studied using the cross-streaking technique. Of 271 meningococcal isolates tested, > 84% were sensitive to this strain. The degree of susceptibility was largely dependent upon the agent characteristics of the individual isolates. Serogroup A sulfonamide-resistant systemic strains and non-groupable sulfonamide-sensitive isolates from healthy carriers were highly sensitive to the antagonistic activity. Among insensitive or weakly sensitive strains, serogroup B sulfonamide-resistant isolates dominated. The activity is of general interest as it also antagonized growth of bacteriocin producers. Colonization by the producer strain might determine the agent characteristics of a surviving population. Group B was predominant among disease-causing strains in Norway at the time when strain 77/79A was isolated. A component was purified by ammonium sulfate precipitation, gel filtration and hydrophobic interaction chromatography. It was bacteriostatic and partly resistant to proteolysis by trypsin. Preparations remained active after 30 min at 90 degrees C, but activity was lost after 20 min at 120 degrees C. Nevertheless, sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced a band by Coomassie Brilliant Blue staining, corresponding to a molecular mass of approximately 52 kDa. Further characterization was limited due to the low levels of active substance produced.

Production, isolation and purification of bacteriocins expressed by two strains of Neisseria meningitidis.

The systemic Neisseria meningitidis strain P241 and the healthy pharyngeal carrier strain BT878 produce bacteriocin-like substances during growth. A method has been devised for obtaining the active substances in solution. The activity was recovered by freeze-thaw extraction of dialyzed Todd-Hewitt agar medium into which the bacteriocins had diffused during growth of the producer strains. The bacteriocins were purified more than 50-fold by ammonium-sulphate precipitation and hydrophobic interaction chromatography. They are quite stable to heat and remain active 100% after 30 min at 100 degrees C. However, the protein nature of the bacteriocins has been confirmed by their sensitivity to alpha-chymotrypsin. Gel filtration indicated an Mr of 100-110 kDa, whereas SDS-polyacrylamide gel electrophoresis produced a common band by Coomassie staining corresponding to an Mr of 47-48 kDa, suggesting a dimer form of the active protein component.

Bacteriocins in Neisseria meningitidis. Screening of systemic patient strains and pharyngeal isolates from healthy carriers.

Systemic meningococcal isolates and meningococci from healthy pharyngeal carriers in Norway were screened for production of growth antagonistic substances. Seven (4.9%) of a total of 142 systemic strains and 3 (2.1%) of 140 carrier isolates spontaneously released diffusible growth antagonistic substances. Properties shown by these substances complied with the criteria used in the definition of a bacteriocin. A cluster of producers among systemic strains registered during the first half of 1975 in North Norway (13.5% of the isolates) was observed, coinciding with the peak in incidence of meningococcal disease of the Norwegian epidemic starting in that region. Among more recent isolates, producers occurred at approximately the same rate in systemic strains (2.5%) as in carrier isolates. The meningocin-producing isolates detected were either of serogroup A and generally sulfonamide-resistant, or serogroup B and sulfonamide-sensitive. The group A strains isolated from disease cases in North Norway during the first half of 1975 were mostly sulfonamide-resistant. Except for the producers, all these strains revealed distinctly higher sensitivity to meningocin than did serogroup B sulfonamide-resistant strains, which became predominant among meningococci causing disease in Norway from that time on.

Inhibitory spectrum of bacteriocin-like agents from Neisseria meningitidis.

Bacteriocin-like agents from five strains of Neisseria meningitidis were active against other meningococci and some other Neisseria species. Meningococci belonging to the same serogroup or serotype could be subdivided into distinct bacteriocin types. Insensitivity to the inhibitory agents was observed more frequently among serologically groupable strains than among non-groupable. Strains belonging to serogroup B were more often insensitive to the inhibitors released by four of the donor strains (P201, P213, P241, 99/79) than other groupable strains. Insensitivity to the bacteriocin-like activity of the fifth donor strain (77/79A) seemed to be evenly distributed among strains of different groups. Strains of serotype 15 were more often insensitive to the five inhibitors than strains belonging to other types. Group B and type 15 are most frequently observed among strains isolated from clinical cases in Norway.

Activity of bacteriocin-like agents from Neisseria meningitidis on strains isolated from patients and carriers.

Strains of Neisseria meningitidis isolated from patients were more often completely insensitive to five bacteriocin-like agents than carrier strains, but no particular bacteriocin type could be associated with patient strains. Two genetically distinct types of insensitivity could be separated by transformation. Insensitivity to four bactericidal agents (from the strains P201, P213, P241 and 99/79) was caused by one common genetic marker whereas insensitivity to a bacteriostatic agent (from the strain 77/79) was caused by another. Insensitivity to the bactericidal agents was more often found among strains belonging to serogroup B than among strains belonging to other groups. But group B strains from patients were more often insensitive than similar strains from carriers. Insensitivity to the bacteriostatic agent seemed evenly distributed among strains of various serogroups. All insensitive patient strains tested showed reduced binding/destruction of inhibitor, indicating that resistance and not tolerance is associated with patient strains.

Influence of blood cells on the expression of bacteriocin-like activity in Neisseria meningitidis.

The inhibitors responsible for the bactericidal activity found in four strains of Neisseria meningitidis were strongly bound to or inactivated by blood cells. The bactericidal effects were specifically influenced by human cells of types O, A, B and AB. Cells from horse, sheep and rabbit had no significant effect. The bacteriostatic effect observed in one strain of N. meningitidis was more moderately reduced in the presence of animal cells as well as human cells. The effects of the blood cells were connected with pellets from lysates of the blood cells. The bacteriostatic effect was also moderately reduced in the presence of plasma, hemoglobin and methemoglobin regardless of origin, and to some extent of cytochrome C. CaCl2 slightly enhanced the bacteriostatic effect.

Expression of bacteriocin-like activity in batch cultures of Neisseria meningitidis and consequences for genetic recombination.

The bactericidal activity found in four strains of Neisseria meningitidis and the bacteriostatic effect observed in one were expressed in the exponential phase of batch cultures and continued during the stationary phase. The active material was stable for more than 50 hours in agar media. In mixed cultures a strain with inhibitory activity eradicated a sensitive strain, and dominated the culture in the latter part of the exponential phase. Recombination took place in mixed cultures when at least one of the strains had the genetic properties which determine competence in transformation. The presence of inhibitory activity increased the recombination frequency at least ten-fold. Genetic markers were introduced into a strain with inhibitory activity from sensitive strains of N. meningitidis and from N. cinerea.

Three types of growth inhibition among strains of Neisseria meningitidis isolated from patients in Norway.

A survey has been made of Neisseria meningitidis strains isolated from patients in order to determine the prevalence of diffusible growth inhibitory substances active against other meningococci. Three types of growth inhibitory activity could be distinguished. The first type was release in cultures of nearly all strains after treatment with chloroform vapour. This activity inhibited all meningococci tested, including the donor strains. The properties were similar to those of the non-specific growth inhibitors observed among most strains of N. gonorrhoeae. The second type was demonstrated in cultures from four strains among 215 strains tested and may be present in a fifth strain. Bactericidal activity with high strain specificity was spontaneously released from cultures of the four strains. This type of activity was found to fit the description of a bacteriocin. The third type of inhibition was observed in one strain among the 215 strains tested. Cultures of this strain spontaneously released bacteriostatic activity. Products from the strain could inhibit almost all meningococcal strains except the donor strain itself. This activity was not entirely blocked by bovine serum albumin; it was not abolished by pronase and it was not stopped by dialysis membranes with cut-off 12000.