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.

Fastidious gram-negative bacteria: meeting the diagnostic challenge with nucleic acid analysis.

The extended panorama of fastidious Gram-negative bacteria (FGNB) as opportunistic etiological agents of infectious diseases in immunocompromised patients is largely due to improved medical expertise and technology. The heightened awareness of infectious diseases due to FGNB species mandates comprehensive classification and identification systems as a basis for rapid and reliable diagnostics. The most useful approaches are combinations of nucleic acid techniques such as hybridization, genetic transformation, amplification and base sequence analysis with selected conventional criteria. Among these approaches, the widely distributed feature of natural competence in these organisms facilitates the use of the biological method of genetic transformation as a valuable addition to the more common nucleic acid techniques. We describe the development of the taxonomy of FGNB through the last four decades, with particular emphasis on the families Neisseriaceae, Moraxellaceae, and Pasteurellaceae.

Moraxella caprae sp. nov., a new member of the classical Moraxellae with very close affinity to Moraxella bovis.

Eight phenotypically homogeneous Moraxella-like strains were isolated from the nasal flora of healthy goats. Total genomic DNA-DNA hybridization, DNA base composition determination, and genetic transformation studies were performed to determine the relationships of these bacteria to the classical moraxellae. The eight new isolates exhibited very high levels of genetic affinity to Moraxella bovis, as shown by quantitative and qualitative genetic transformation data, and exhibited high DNA-DNA relative binding ratios to each other (63% or more) but lower levels of DNA homology with all of the other species investigated, including the closely related classical moraxellae. Our results, combined with the general morphologic and phenotypic profiles of these organisms, indicate that they should be classified with the classical moraxellae, and we propose the name Moraxella caprae for them. Strain 8897 (= CCUG 33296 [corrected] = NCTC 12877) is the type strain of M. caprae.

A polysaccharide produced by a mucoid strain of Moraxella nonliquefaciens with a 2-acetamido-2-deoxy-5-O-(3-deoxy-beta-D-manno-octulopyranosyl)-beta-D- galactopyranosyl repeating unit.

A capsular polysaccharide, isolated from the mucoid Moraxella nonliquefaciens strain 3828/60, has been investigated by component analyses, periodate oxidation, methylation analyses, mass spectrometry, 1H and 13C NMR spectroscopy, and hydrolysis to give a disaccharide that was isolated and characterised. The results showed that the polysaccharide has the repeating unit-->3)-beta-D- GalpNAc-(1-->5)-beta-Kdo p-(2-->, with approximately 40% of O-8 of Kdo being acetylated.

Sequence divergence in two tandemly located pilin genes of Eikenella corrodens.

Eikenella corrodens normally inhabits the human respiratory and gastrointestinal tracts but is frequently the cause of abscesses at various sites. Using the N-terminal portion of the Moraxella nonliquefaciens pilin gene as a hybridization probe, we cloned two tandemly located pilin genes of E. corrodens 31745, ecpC and ecpD, and expressed the two pilin genes separately in Escherichia coli. A comparison of the predicted amino acid sequences of E. corrodens 31745 EcpC and EcpD revealed considerable divergence between the sequences of these two pilins and even less similarity to EcpA and EcpB of E. corrodens type strain ATCC 23834. EcpC from E. corrodens 31745 displayed high degrees of homology to the pilins of Neisseria gonorrhoeae and Pseudomonas aeruginosa. EcpD from E. corrodens 31745 showed the highest homologies with the pilin of one of the three P. aeruginosa classes, whereas EcpA and EcpB of strain ATCC 23834 most closely resemble Moraxella bovis pilins. These findings raise interesting questions about potential genetic transfer between different bacterial species, as opposed to convergent evolution.

Differentiation of Moraxella nonliquefaciens, M. lacunata, and M. bovis by using multilocus enzyme electrophoresis and hybridization with pilin-specific DNA probes.

Genetic relationships among strains of Moraxella nonliquefaciens, M. lacunata, and M. bovis were studied by using multilocus enzyme electrophoresis and DNA-DNA hybridization. The 74 isolates analyzed for electrophoretic variation at 12 enzyme loci were assigned to 59 multilocus genotypes. The multilocus genotypes were grouped in four major clusters, one representing strains of M. nonliquefaciens, two representing strains of M. lacunata, and one comprising strains of M. bovis and the single strain of M. equi analyzed. DNA-DNA hybridization with total genomic probes also revealed four major distinctive entities that corresponded to those identified by multilocus enzyme electrophoresis. The two distinct clusters recognized among the M. lacunata strains apparently corresponded to the species previously designated M. lacunata and M. liquefaciens. Distinction of the four entities was improved by hybridization with polymerase chain reaction products of nonconserved parts of pilin genes as DNA probes. With these polymerase chain reaction probes, new isolates of M. nonliquefaciens, M. lacunata, M. liquefaciens, and M. bovis can be identified easily by hybridization.

The type 4 pilin of Moraxella nonliquefaciens exhibits unique similarities with the pilins of Neisseria gonorrhoeae and Dichelobacter (Bacteroides) nodosus.

Moraxella nonliquefaciens is a bacterium which is part of the normal flora of the human upper respiratory tract and is an occasional cause of disease. Using a previously cloned type 4 pilin gene (tfpQ) from Moraxella bovis as a hybridization probe, we have cloned an 826 bp Sau3 AI fragment which contains an M. nonliquefaciens type 4 pilin gene (tfpA) from strain NCTC 7784. The pilin gene is expressed in Escherichia coli. We have examined NCTC 7784 and nine other M. nonliquefaciens strains by genomic Southern hybridization using tfpA as a probe, and they all appeared to have more than one pilin gene. While the predicted amino acid sequence of the M. nonliquefaciens tfpA pilin has conserved regions as compared to pilins of M. bovis and M. lacunata, it also shows similarities to both the type 4 pilin of Neisseria gonorrhoeae and the type 4 pilin of Dichelobacter nodosus (formerly Bacteroides nodosus).

Some arguments on early hospital admission and treatment of suspected meningococcal disease cases.

Arguments are presented which indicate or show that: (1) Diagnostic precision and severity level of systemic meningococcal disease (MCd) both seem to rise exponentially with the developmental stage of the disease at referral. Lowering of the clinical admission threshold improves early coverage of vaguely suspected cases and should probably be implemented in Norway. (2) Fear that cell wall active bactericidal antibiotics could trigger important endotoxin release may cause unnecessary treatment delays. (3) Although risk of death due to meningococcal septicemia is the main indication for early treatment of MCd, the risk of sequelae may also become a major cause for very early treatment of MCd. (4) Treatment delays for MCd cases could have been substantially reduced by implementing reasonable guidelines. (5) In the relations between the public and the health service both organizational and psychological factors are operating. (6) Increased awareness among professionals and lay people of some key symptoms and signs may facilitate earlier diagnosis of MCd. (7) Earlier treatment of meningococcal disease is now feasible and does seldom preclude the possibility of etiological diagnosis. (8) More relevant studies and information on the early phases and rapid management of MCd are highly desirable. Rapid diagnosis and treatment of MCd are very important to reduce death, sequelae and community costs and should be more advocated in training of health personnel and in public information.

Identification of Haemophilus aphrophilus and Actinobacillus actinomycetemcomitans by DNA-DNA hybridization and genetic transformation.

DNA-DNA hybridization was used to identify clinical isolates as Haemophilus aphrophilus or Actinobacillus actinomycetemcomitans. Some of the isolates were naturally competent for genetic transformation and were also used as DNA recipients for identification of other isolates. The results obtained by hybridization were supported by interstrain-to-intrastrain transformation ratios. Distinction between the closely related species H. aphrophilus and A. actinomycetemcomitans was generally clear-cut by both methods. Distinction of H. aphrophilus and A. actinomycetemcomitans from type and reference strains of a diversity of species in the family Neisseriaceae and other gram-negative species was also demonstrated by both methods. This is the first description of the identification of clinical isolates of H. aphrophilus or A. actinomycetemcomitans by using them as recipients in genetic transformation. The results suggest that this is a reliable system for identification of new clinical isolates belonging to these taxonomic entities.

Neisseria meningitidis group B capsular polysaccharide. Bacterial content and release in relation to categories of infection and filtrability of released endotoxin.

A total of 41 Neissera meningitidis isolates were analyzed for capsular polysaccharide (CP) and lipopolysaccharide (LPS) release and filtrability. Twenty-two of these isolates were serogroup B from blood or cerebrospinal fluid of patients, five were group B isolates from healthy throat carriers, and 14 were nongroupable (acapsular) meningococcal isolates from healthy carriers. Filtration of liquid whole-cell cultures through cellulose acetate-nitrate filters resulted in distinctly lower LPS filtrate activity for acapsular than for capsular meningococci (p less than 0.001). On the other hand, when polysulfone membrane filtration was performed, filtrates from acapsular and capsular meningococci contained LPS in similar amounts. These results indicate that LPS-containing particles released from acapsular isolates are larger or more aggregated than corresponding CP- and LPS-containing particles released from capsular isolates. The LPS released from acapsular isolates apparently are more efficiently retained by adsorption to cellulose acetate-nitrate. From the capsular isolates comparatively more CP than LPS appeared to be released, as related to cell-bound amounts. The total amounts of CP and released, filtrable LPS through cellulose acetate-nitrate filters were both relatively low and had similar values for capsular carrier meningococci and systemic isolates from mild meningococcal disease. The amount of CP released and passing this type of membrane was significantly higher for systemic isolates from severe septicemia than from mild meningococcal disease (p = 0.03).

A unique plasmid profile characterizing Salmonella enteritidis isolates from patients and employees in a hospital.

Plasmid profiling was used as an epidemiological tool during a period of frequent Salmonella enteritidis infection in a hospital. S. enteritidis was isolated from 22 patients and employees. Isolates from 18 persons harbored one 29 and one 36 megadalton (MDa) plasmid. The 29 MDa plasmid has not been previously described in this species and was not found in 54 control strains of S. enteritidis from other sources. The respective restriction endonuclease digest fragments of the 36 and the 29 MDa plasmids were always identical. This plasmid pattern thus served as a marker for the isolates from the outbreak.

Sulfonamide resistance in Neisseria meningitidis isolates of clones of the ET-5 complex.

A distinctive group of genetically closely related clones, as determined by multilocus enzyme electrophoresis, the ET-5 complex, has been responsible for an epidemic of meningococcal disease in Norway since the mid-1970's. Most isolates of the ET-5 complex from Norway are sulfonamide-resistant, serogroup B, and serotype 15:P1.16. Clones of the ET-5 complex that have been identified as the causative agents of recent outbreaks and epidemics in many other parts of the world show, outside Northern Europe, different associations of serotype protein antigens. We here report the analysis of sulfonamide susceptibility of isolates of the ET-5 complex from various geographic sources. There was no difference in resistance according to geographic source, serogroup, or serotype of the isolates, demonstrating that, in contrast to serotype and serogroup, sulfonamide resistance is an essentially invariant property of clones of the ET-5 complex.

DNA-DNA hybridization incompatibility of Campylobacter pylori with other Campylobacter and Wolinella species.

DNA-DNA hybridization in solution was used to characterize 23 human isolates of Campylobacter pylori. The 23 isolates showed DNA affinity with the type strain (NCTC 11637). The relative binding ratios varied between 0.83 and 1. Type strains of C. coli (NCTC 11366), C. jejuni (NCTC 11351), C. laridis (NCTC 11352), C. sputorum subsp. sputorum (ATCC 35980), Wolinella recta (NCTC 11489) and W. succinogenes (ATCC 29543) showed relative binding ratios less than 0.01 compared to the C. pylori type strain. The results suggest that C. pylori is a homogenous taxonomic unit distinctly separated from these other species.

Differentiation of some species of Neisseriaceae and other bacterial groups by DNA-DNA hybridization.

DNA-DNA hybridization using total genomic DNA probes may represent a way of differentiating between miscellaneous bacterial species. This was studied with type and reference strains of 20 species in Moraxella, Kingella, and other selected Gram-negative groups. Both radioactive and biotin labelling were employed. Most of the species examined were easily distinguished, such as Moraxella (Branhamella) catarrhalis, M.(B.) ovis, M. atlantae, M. phenylpyruvica, M. osloensis, Neisseria elongata, N. meningitidis, Kingella kingae, K. indologenes, K. dentrificans, Oligella urethralis, Eikenella corrodens, Cardiobacterium hominis, Haemophilus aphrophilus, Actinobacillus actinomycetemcomitans, Gardnerella vaginalis, and DF-2. This reflected the extent of the genetic distances between them as a basis for identification by hybridization. There was some clustering in the Moraxella group. Especially the closely related Moraxella nonliquefaciens, M. lacunata and M. bovis showed strong hybridization affinities. This leads to potential problems in distinguishing these three species from each other by DNA-DNA hybridization with total genomic probes alone.

Meningococcal phenotypic and genotypic characteristics and human antibody levels.

During a 2-month period in 1984, throat and blood samples were collected from 1,102 healthy persons of different ages living in the city of Tromsø, Norway. One hundred and eight persons (9.8%) were meningococcal carriers, but the carrier rate varied with sex and age. Twenty-nine isolates (26.9%) were of serogroup B, and 31 (28.7%) isolates contained the serotype 15 antigen. Sixty-eight (63.0%) isolates were nontypable, 49 (45.4%) were nongroupable, and 21 (19.4%) were sulfonamide resistant. All nine serotype 2a isolates and eight (25.8%) of the serotype 15 isolates were sulfonamide resistant. Only these eight serotype 15, sulfonamide-resistant isolates had a DNA fingerprint similar to that of the majority of systemic isolates of Neisseria meningitidis in Norway. The average level of antimeningococcal immunoglobulin G antibodies, as determined by a whole-bacterium enzyme immunoassay with a systemic B:15 meningococcal strain as the antigen, was low until 12 to 15 years of age and then steadily increased.