David Hansman

Increased Resistance to Penicillin of Pneumococci Isolated from Man

Abstract Strains of Diplococcus pneumoniae relatively insensitive to penicillin were isolated from an aboriginal child in Australia and from 15 New Guineans. The concentration of penicillin required to inhibit growth was 25 times that which inhibited sensitive pneumococci. These pneumococci were also partially resistant to cephalosporin antibiotics. All the relatively insensitive pneumococci from New Guinea were identified as Type 4, suggesting that transmission of the partially resistant strain had occurred. The isolations were made from persons who lived in remote areas where penicillin had frequently been used.

Comparative efficacy of pneumococcal neuraminidase and pneumolysin as immunogens protective against Streptococcus pneumoniae

Neuraminidase and pneumolysin were purified from cultures of Streptococcus pneumoniae and used, either singly or in combination, to immunize juvenile mice which were subsequently challenged intranasally with virulent S. pneumoniae. In each of two independent trials, a small but significant (P less than 0.05) increase in survival time (compared with that of non-immunized mice) was observed in groups which had been immunized with neuraminidase, but only if the enzyme had been pre-treated with 3.4% (v/v) formaldehyde. The median extension in survival time was significantly less (P less than 0.01) than that of mice which had been immunized with pneumolysin alone. The median survival time for mice which had received both formaldehyde-treated neuraminidase and pneumolysin was not significantly different from that of mice which had received pneumolysin alone. While these findings provide direct evidence that neuraminidase contributes to the pathogenicity of the pneumococcus in mice, they suggest that this protein may be of less value than pneumolysin as a vaccine component in the present experimental model.

Comparative efficacy of autolysin and pneumolysin as immunogens protecting mice against infection by Streptococcus pneumoniae

Previous studies on Streptococcus pneumoniae have established that the pneumococcal proteins autolysin (N-acetylmuramyl-L-alanine amidase) and pneumolysin both contribute significantly to the virulence of the organism. In the present work, autolysin and a defined toxoid derivative of pneumolysin were tested, individually and in combination, for efficacy in a mouse model as antigens protecting against challenge with virulent, wild-type S. pneumoniae. While each antigen alone provided significant protection, the degree of protection was not increased when the antigens were administered together. In an additional experiment, mice were challenged with a genetically-modified mutant strain of pneumococcus unable to express active pneumolysin. Pre-immunization of such mice with autolysin failed to provide any significant protection against the challenge. The results of this study suggest that the most important contribution made by autolysin to the virulence of S. pneumoniae may be its role in mediating the release of pneumolysin from the pneumococcal cytoplasm during infection.

Effect of insertional inactivation of the genes encoding pneumolysin and autolysin on the virulence of Streptococcus pneumoniae type 3

Derivatives of Streptococcus pneumoniae type 3 deficient in production of either pneumolysin or autolysin were constructed. This was achieved by transformation of type 3 pneumococci with DNA from derivatives of a rough strain (Rx1), in which the respective genes had been interrupted by insertion-duplication mutagenesis using internal fragments of the cloned genes in the vector pVA891. Southern blot analysis confirmed that the pneumolysin or autolysin genes in the respective transformants had been interrupted by insertion of the plasmid-derived sequences. Both the pneumolysin-negative and the autolysin-negative strains had significantly reduced (P less than 0.0001) virulence in mice, as judged by survival time after intraperitoneal challenge. The median survival time of mice challenged with type 3 pneumococci in which either pneumolysin or autolysin production had been reconstituted by back-transformation of the mutants with an intact copy of the respective cloned gene (with concomitant elimination of plasmid-derived sequences), was indistinguishable from that of mice challenged with the wild-type strain. These results establish the importance of both pneumolysin and autolysin to the virulence of type 3 pneumococci.

Purification and immunological characterization of neuraminidase produced by Streptococcus pneumoniae

Previous workers have suggested that Streptococcus pneumoniae, the pneumococcus, produces multiple forms of the enzyme neuraminidase. By serial chromatography on DEAE-cellulose, Sephacryl S-200, Amicon Red-A gel and hydroxylapatite we have purified to electrophoretic homogeneity a pneumococcal neuraminidase with an apparent molecular weight of 86,000 (as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis). Mouse antiserum raised against the purified material reacted with a single species with molecular weight 107,000 (107K form) in crude pneumococcal cell lysate. During the purification procedure this species was progressively degraded to the molecular weight 86,000 (86K) form whilst retaining enzyme activity. Degradation of neuraminidase was inhibited by phenylmethylsulphonylfluoride (PMSF) and ethylenediaminetetraacetic acid (EDTA). Purification of the enzyme in the presence of these protease inhibitors permitted the isolation of the 107K species substantially undegraded and greater than 98% pure. Our findings on the degradation of neuraminidase during its purification account for previous reports of multiple neuraminidase isoenzymes in Streptococcus pneumoniae.

High prevalence of penicillin-insensitive pneumococci in Port Moresby, Papua New Guinea.

One-third of 57 strains of pneumococci isolated from patients with severe pneumococcal infections in Port Moresby, Papua New Guinea, in 1978 were penicillin-insensitive (PR). The infections were community-acquired and comprised cases of bacteraemia, bacteraemic pneumonia, and meningitis. The prevalence of invasive PR strains in children was significantly greater than in adults. Twenty-one serotypes were identified, of which ten types (6, 9, 10, 12-14, 18, 19, 23, 24) showed diminished sensitivity to penicillin; with types 12 and 18 penicillin-insensitivity has not been met with previously in Papua New Guinea and only rarely elsewhere; seven of the 19 PR strains belonged to type 6. Amongst insensitive strains, minimum inhibitory concentrations of penicillin G ranged from 0.1 to 1.0 microgram/ml (resistance ratios 5 to 50). Resistance to chloramphenicol and other antimicrobial drugs was not encountered.

Protection of infant mice from challenge with Streptococcus pneumoniae type 19F by immunization with a type 19F polysaccharide-pneumolysoid conjugate

The immunogenicity and protective efficacy of a conjugate of Streptococcus pneumoniae type 19F polysaccharide and a genetically toxoided derivative of the pneumococcal toxin pneumolysin was investigated in an infant mouse model. The conjugate was administered to Balb/c mice during pregnancy and/or lactation, and to their offspring during early infancy. The anti-polysaccharide and anti-pneumolysin titres of the immunized infant mice were significantly higher than those of non-immunized controls. When the infant mice were challenged with type 19F pneumococci, the bacteria were cleared more effectively from the blood of immunized mice than from that of control mice. The survival rate for the immunized mice was also significantly higher than that for the control group. These results indicate that highly protective anti-pneumococcal responses can be induced in infant mice by immunization with the conjugate during gestation or early infancy, and suggest a possible role for pneumolysoid-polysaccharide conjugates as human vaccine components.

Control of group C meningococcal disease in Australian Aboriginal children by mass rifampicin chemoprophylaxis and vaccination

An outbreak of 12 cases of meningitis, 11 caused by Neisseria meningitidis serogroup C, occurred at Doomadgee from September, 1990, to April, 1991. The incidence of meningitis was 17.55/10(3) person-years. Only children aged 1-10 years were affected. In October, 1990, or shortly thereafter, 473/509 children aged between 1 and 15 years inclusive had one dose of Mencevax AC. From the time of vaccination until April, 1991, a further eight cases occurred, six in vaccinated children. Vaccine efficacy in 1-15 year olds was calculated as 77%. Despite this, in April, 1991, the prevalence of antibody to group C polysaccharide in vaccinated children (78%) was not significantly different from that in unvaccinated children and adults. 46 nonresponders were revaccinated, and, in February, 1992, 78% had antibodies to group C polysaccharide. In April, 1991, an estimated 3.0% of the population had group C organisms, carriage being directly related to household crowding. In June, 1991, 2 months after mass prophylaxis with rifampicin, none of these individuals were carriers. In October, 1991, the carriage rate of group C organisms was 0.64%. There have been no further cases caused by the epidemic strain. Although uncrowded housing is a basic need, mass chemoprophylaxis and two doses of vaccine for children should be used in similar outbreaks.

Surveillance of antibiotic resistance in invasive isolates of neisseria meningitidis in Australia 1994–1999

Summary A total of 1434 strains of Neisseria meningitidis isolated from cases of invasive meningococcal disease (IMD) in Australia between 1994 and 1999 were examined by standard methods for susceptibility to antibiotics used for treatment and prophylaxis. The proportion of isolates fully susceptible to penicillin decreased from 45% in 1994 to 26% in 1999 ( P < 0.001). All the other isolates were less sensitive to penicillin except for two meningococci with a penicillin MIC of 1 mg/l. The geometric mean penicillin MIC increased from 0.045 to 0.065 mg/l from 1994 to 1999. There was no significant difference in the geometric mean penicillin MICs of serogroup B and serogroup C meningococci. Penicillin susceptibility was significantly associated with a poorer outcome. Isolates from survivors of IMD had a higher geometric mean penicillin MIC (0.06 mg/l) than those from fatal cases (0.048 mg/l) ( P < 0.001). This suggests that factors other than the decrease in susceptibility to penicillin observed were more relevant to outcome in IMD. All isolates were fully susceptible to ceftriaxone. Rifampicin resistance was infrequent (eight isolates in 6 years) and sporadic. A single isolate had decreased quinolone susceptibility. Despite the significant shift in susceptibility to penicillin recorded, this group of antibiotics remains a suitable treatment for IMD in Australia.

Pneumococcal carriage amongst children in Adelaide, South Australia

Amongst 1267 healthy children 6 months to 4.5 years of age in Adelaide, the pneumococcal carriage rate from a single nasal swab sampling was 29% in the period 1980-1. Of 269 children, sampled monthly on five occasions, 91% carried a pneumococcus on one or more occasions: 55% carried a single type, 33% carried two types, 2% carried three types and 1% carried four types; 18% carried a pneumococcus on either 4 or 5 occasions. The commonest types encountered were types 6, 19 and 23 in that order, and these three types constituted 57% of the total: other common types (greater than 5% of the total) were types 14, 15 and 11, and the six commonest types constituted 77% of the total. Of these, types 6, 14, 19 and 23 commonly cause systemic disease in children; on the other hand types 11 and 15 cause disease infrequently. The number of strains showing antimicrobial drug resistance was low: on quantitative testing 0.7% of 291 isolates examined showed relative resistance to benzylpenicillin and 0.7% were resistant to tetracycline; 10.9% of 230 isolates examined showed resistance to co-trimoxazole; dual or multiple drug resistance was not detected, and all isolates tested were susceptible to chloramphenicol, erythromycin, lincomycin and rifampicin.