Neisseria Strains Research Articles

Butyrate esterase (4-methylumbelliferyl butyrate) spot test, a simple method for immediate identification of Moraxella (Branhamella) catarrhalis [corrected

A total of 47 Moraxella (Branhamella) catarrhalis strains, 89 strains of Neisseria spp., and 82 strains of miscellaneous bacteria and yeasts were studied by using a fluorogenic spot method which detects butyrate esterase. A positive butyrate esterase spot test correctly differentiated M. catarrhalis from Neisseria spp., which had a negative butyrate esterase spot test reaction. The test is rapid, simple, and easy to perform. The butyrate esterase spot test was useful for direct identification of M. catarrhalis from primary cultures. However, false-positive reactions may occur with mixed cultures.

Analysis of the expression of outer-membrane proteins in Neisseria meningitidis in iron-replete and iron-deficient media

A total of 103 Neisseria strains, including 42 carrier and 40 invasive N. meningitidis and 21 commensal species were studied. Outer-membrane proteins from carrier and invasive N. meningitidis showed similar molecular mass distributions (except in the range from 78 to 82 kDa in which 90% of the invasive but only 47.6% of the carrier strains expressed proteins), many strains showin neither class II (41 kDa) nor class III (38 kDa) proteins. When grown in iron-restricted conditions proteins were induced mainly in the ranges from 62 to 92 kDa with no significant differences between groups. Commensal species, both in normal and in iron-restricted conditions, showed outer-membrane protein patterns different from those of N. meningitidis in several molecular mass ranges. Cluster analysis based on expression of principal outer-membrane proteins allowed the differentiation of commensal species and N. meningitidis, although not that of the invasive and carrier groups within the latter.

Erythrocyte gangliosides act as receptors for Neisseria subflava: identification of the Sia-1 adhesin

Neisseria gonorrhoeae was recently shown to bind to a subset of lactose-containing glycolipids (N. Strömberg, C. Deal, G. Nyberg, S. Normark, M. So, and K.-A. Karlsson, Proc. Natl. Acad. Sci. USA 85:4902-4906, 1988). A number of commensal Neisseria strains were also shown to be lactose binders. In addition, Neisseria subflava bound to immobilized gangliosides, such as hematoside and sialosyl paragloboside, carrying the NeuAc alpha 2-3Gal beta 1-4Glc sequence. To a lesser extent, N. gonorrhoeae also bound to this receptor in vitro. In N. subflava GN01, this binding property mediated agglutination of human erythrocytes in a neuraminidase-sensitive fashion. Nitrosoguanidine-induced nonhemagglutinative mutants of N. subflava GN01 had lost the ability to bind hematoside and sialosylparagloboside but remained able to bind lactosylceramide and gangliotetraosylceramide. These mutants fell into three classes with respect to their outer membrane protein profiles in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Class 1 mutants were identical to the parent strain save for the loss of a 27-kilodalton (kDa) protein. Class 2 mutants showed an outer membrane protein profile identical to that of the wild type, whereas mutants belonging to class 3 showed a number of changes, including the apparent absence of the 27-kDa protein. The 27-kDa protein from N. subflava GN01 was purified from the supernatant. A polyclonal antiserum to the purified Sia-1 protein as well as a Sia-1-specific monoclonal antibody inhibited hemagglutination by strain GN01. The purified Sia-1 protein in the presence of diluted anti-Sia-1 antiserum mediated a neuraminidase-sensitive hemagglutination. The purified Sia protein from a class 2 mutant was not able to hemagglutinate when cross-linked with antibodies, suggesting that it is a mutant form of Sia-1 affected in the receptor-binding site. Immunoelectron microscopy with a Sia-1-specific monoclonal antibody revealed that the adhesin was nonfimbrial in nature, with aggregates of the adhesin extended out from the cells in a patchy fashion.

Expression of carbonic anhydrase in neisseriae and other heterotrophic bacteria

A diverse range of heterotrophic bacteria was screened for the presence of carbonic anhydrase (CA) activity, sensitivity to inhibition of growth by acetazolamide (CA inhibitor), and the presence of protein binding monospecific antibody prepared against purified Neisseria sicca CA. CA activity was demonstrated only in strains of N. sicca and N. gonorrhoeae. However, all Neisseria strains, including various isolates of N. meningitidis and N. lactamica, were sensitive to acetazolamide, when grown in air, and showed serological cross-reaction with N. sicca CA. Strains of other genera were resistant to acetazolamide. A number of strains including members of the genera Pseudomonas, Staphylococcus, Streptococcus, Serratia and Proteus also strongly expressed a gene product(s) immunologically related to CA. The presence of CA cross-reacting proteins, which lack hydrase activity, is discussed in relation to the function of the various mammalian CA isoenzymes.

Evaluation of five rapid systems for the identification of Neisseria gonorrhoeae

Five commercial kit systems for the rapid identification of Neisseria gonorrhoeae were evaluated. The systems were tested with various pathogenic and saprophytic Neisseria and Branhamella strains having reactions typical for their individual species. Three systems (RIM-N, quadFERM, and RapID NH) were found to be 100% sensitive and specific.

In vitro evaluation of WIN 57273, a new broad-spectrum fluoroquinolone

WIN 57273 is a new fluoroquinolone that has an expanded spectrum of activity against Staphylococcus spp. (MIC for 90% of isolates [MIC90], 0.008 microgram/ml), Enterococcus faecalis (MIC90, 0.06 microgram/ml), Bacillus spp. (MIC90, 0.03 micrograms/ml), Listeria monocytogenes (MIC90, 0.06 microgram/ml), Streptococcus spp. (MIC90, 0.03 microgram/ml), and Bacteroides fragilis group strains (MIC90, 0.5 microgram/ml). Like other fluoroquinolone compounds, WIN 57273 was active against members of the family Enterobacteriaceae (97% of strains inhibited by less than or equal to 2 micrograms/ml), Haemophilus, Branhamella, and Neisseria strains (100% susceptible), Acinetobacter spp. (100% susceptible), and Pseudomonas aeruginosa (68% susceptible). We observed that WIN 57273 was very active against cephalosporin- or aminoglycoside-resistant gram-negative strains but shared cross-resistance with other fluoroquinolones. Increasing inoculum concentrations had minimal effects on WIN 57273 MICs, and the drug was considered to be bactericidal based on reference MBC and kill curve analyses. Unlike most previously studied drugs in this class, WIN 57273 had increased activity (three- to fourfold) at low pH. Rates of mutation to WIN 57273 resistance at eight times its MIC were in the range of 5.6 x 10(-8) to greater than 1.4 x 10(-9). This new compound possesses a wide potential spectrum of use, and it should be evaluated further by in vitro and in vivo studies.

In-vitro activity of sparfloxacin, a new quinolone antimicrobial agent

The in-vitro activity of sparfloxacin (AT-4140), a new difluorinated quinolone, was compared with those of ciprofloxacin, temafloxacin and selected members of other groups of antimicrobial agents, against 651 recent distinct clinical isolates and strains with known mechanisms of resistance. Three strains of Chlamydia trachomatis were also studied. The MICs for 90% of the Enterobacteriaceae were between 0.06 and 1 mg/l; for Pseudomonas aeruginosa the MIC90 was 2 mg/l. Sparfloxacin was 16-fold more active against Acinetobacter spp. than ciprofloxacin. For Staphylococcus spp., Streptococcus, spp. and Enterococcus faecalis the MIC90 was between 0.25 and 1 mg/l; sparfloxacin was four-fold more active against Str. pneumoniae than ciprofloxacin. Ninety percent of strains of Haemophilus influenzae, Branhamella catarrhalis and Neisseria spp. were inhibited by less than 0.03 mg/l; for Bacteroides fragilis the MIC90 was 1 mg/l. The three strains of Chl. trachomatis were susceptible to 0.06-0.12 mg/l sparfloxacin, which was 16-fold more active than ciprofloxacin. There was cross resistance among the quinolones, but not between the quinolones and other groups of antimicrobials. The protein binding of sparfloxacin was 40% and serum had little effect on its activity.

Serum antibodies to cross‐reactive Neisseria outer membrane antigens in healthy persons and patients with meningococcal disease

Outer membranes (OMs) were prepared from the Neisseria meningitidis (Nm) strain 44/76, N. gonorrhoeae (Ng) NRL 8658, and N. lactamica (N1) ATCC 23970. Paired serum samples from 16 patients with serogroup B Nm disease and single samples from 30 blood donors were tested for IgG antibody levels against the three OMs in indirect ELISA, before and after absorption of the sera with N1 OM. Immunoblot analysis was used to identify OM target antigens for cross-reacting and strain-specific antibodies. Most of the Nm- and NG-antibodies in sera from healthy adults were directed against OM antigens shared by the three Neisseria strains. Nm disease induced antibody formation against common Neisseria antigens, identified as the H.8 antigen and LPS determinants, against LPS determinants shared only by the Nm and Ng strains and against a variety of Nm-specific OM antigens. Very low levels of Nm-specific antibodies characterized the Nm patients in the acute phase. Also, the results indicate that the OM ELISA which has been used for the diagnosis of Nm disease, would be more useful if antibodies against common Neisseria antigens were removed from the sera before testing.

Lipooligosaccharides: The Principal Glycolipids of the Neisserial Outer Membrane

The outer-membrane glycolipids of bacteria that colonize mucosal surfaces that are not routinely bathed by bile acids often lack the long, hydrophilic and neutral polysaccharides that protect the lipid membranes of enteric bacteria from dispersal. The glycolipid from these organisms is properly termed a lipooligosaccharide. A Neisseria strain makes from two to six lipooligosaccharide molecules that range in Mr from 3,150 to 7,100. Different species of Neisseria commonly make lipooligosaccharides of identical Mr and epitope content. Differences in oligosaccharides account for most of the observed physical heterogeneity. Oligosaccharides consist of (1) partially conserved and highly substituted basal oligosaccharides that branch at heptose residues; (2) a linear segment consisting of (hexose)n residues that determines the length of the oligosaccharide; and (3) terminal sequences that are similar to those of glycosphingolipids. Epitope expression is linked to physical heterogeneity and is modified by the molecular environment of the outer membrane. Serotype epitopes are present only on lipooligosaccharides of a certain Mr. Certain lipooligosaccharides regulate complement activation onto the bacterial surface and, hence, immune lysis.

High-level tetracycline resistance resulting from TetM in strains of Neisseria spp., Kingella denitrificans, and Eikenella corrodens.

Similar to Neisseria gonorrhoeae, tetracycline-resistant isolates of N. meningitidis, Kingella denitrificans, and Eikenella corrodens contained 25.2-megadalton plasmids carrying the TetM determinant. In contrast, tetracycline-resistant N. subflava biovar perflava-N. sicca and N. mucosa isolates carried the TetM determinant in the chromosome.

The immunochemistry of neisserial LOS

The outer membrane glycolipids of Neisseria lack long polysaccharides and are properly termed lipooligosaccharides (LOS). A Neisseria strain makes from two to six LOS of Mr 3150-7100. Different species commonly make LOS of identical Mr and epitope content. Oligosaccharide (OS) differences account for physical heterogeneity. OS consist of a conserved triantenary basal oligosaccharide, two linear segments of (n) hexose residues that determine OS mass, and terminal sequences similar to those of glycosphyngolipids. Epitope expression is linked to physical heterogeneity and conditioned by the molecular environment of the outer membrane. Serotype epitopes are expressed on Mr-restricted LOS. LOS regulate complement activation onto the bacterial surface and, hence, immune lysis.

Analysis of penicillinase-producing Neisseria gonorrhoeae isolates in Madrid (Spain) from 1983-85.

Between April 1983 and December 1985, 576 strains of Neisseria gonorrhoeae were isolated in our laboratory from patients attending Sexually Transmitted Diseases (STD) clinics. Of these, 61 (10.6%) were penicillinase-producing. Studies on these strains by plasmid analysis, auxotyping and serogrouping showed that the predominant type strains harboured the Asian resistance plasmid, were prototrophic, and were of serogroup W II/W III. About half of the strains, both of the African and Asian type, harboured the transfer plasmid. Strains of serogroup W II/W III were less sensitive to tetracycline and cefoxitin than serogroup W I strains.

Phadebact Monoclonal GC OMNI Test for confirmation of Neisseria gonorrhoeae.

The Phadebact Monoclonal GC OMNI Test (Pharmacia Diagnostics, Piscataway, N.J.) is used for the definitive identification of Neisseria gonorrhoeae. In this test, boiled organisms are examined by using a 1-min coagglutination technique. A total of 776 Neisseria strains, confirmed to the species level by patterns of acid production from carbohydrates incorporated in cysteine-tryptose agar or morphologically consistent with Neisseria meningitidis and fluorescent antibody negative, were tested by the coagglutination technique. Of the 516 isolates of N. gonorrhoeae, 8 (1.6%) were negative with the OMNI Test. Of the 260 isolates of Neisseria spp. other than N. gonorrhoeae, none showed a positive coagglutination reaction. The Phadebact Monoclonal GC OMNI Test provided rapid, accurate identification of N. gonorrhoeae (sensitivity, 98.4%; specificity, 100%).

A note on hydrolysis of tributyrin by Branhamella and Neisseria.

Sixty-three strains of Branhamella and Neisseria were tested by two methods for their ability to hydrolyse glycerol tributyrate. After the conventional plate test, gas liquid chromatographical (GLC) analysis of the agar medium was carried out to detect the hydrolysis product, butyric acid, and other volatile fatty acids. All strains of Branhamella catarrhalis, Neisseria caviae, N. cuniculi and N. ovis but no other Neisseria spp. gave positive results with the conventional test. With GLC, however, most strains of Branhamella and Neisseria were shown to liberate butyric acid. In addition, some strains liberated acetic and isovaleric acids. Greater amounts of butyric acid were produced by clinical strains, in particular B. catarrhalis, compared with reference strains. It was concluded that the conventional plate test for tributyrin hydrolysis differentiates B. catarrhalis, N. caviae, N. cuniculi and N. ovis from other Neisseria.

Branhamella catarrhalis: significance in pulmonary infections and bacteriological features.

A three-month survey revealed 29 patients at our hospital with symptoms of acute pulmonary infection, from whom Branhamella catarrhalis was isolated from lower respiratory tract specimens, in 18 cases in pure culture. Approximately 2% of all respiratory tract specimens examined during the period yielded growth of B. catarrhalis. All except one patient suffered from chronic pulmonary disease, notably chronic bronchitis. A phenotypic comparison was made between 55 strains of B. catarrhalis, of which 50 were recent isolates from lower respiratory tract specimens, and 23 Neisseria strains representing Neisseria meningitidis, Neisseria gonorrhoeae, Neisseria cinerea, Neisseria flavescens, Neisseria mucosa, Neisseria pharyngis, and Neisseria lactamica. The morphology of B. catarrhalis colonies is very characteristic, and when the diagnosis is suspected, testing for the ability to hydrolyze tributyrin may confirm it within hours. Ability to produce deoxyribonuclease is another property which differentiates B. catarrhalis from the Neisseria species. Otherwise, the combination of nitrate reduction and failure to produce acid from glucose, maltose, and sucrose establishes the diagnosis.

Characterization of a beta-lactamase-specifying plasmid isolated from Eikenella corrodens and its relationship to a commensal Neisseria plasmid.

A 9.4-kilobase plasmid encoding penicillin, streptomycin, and sulfonamide resistance was isolated from a beta-lactamase-producing Eikenella corrodens strain. This plasmid appears to be identical to a resistance plasmid common to saprophytic Neisseria strains.

Evaluation of GonoGen coagglutination test for serodiagnosis of Neisseria gonorrhoeae: Identification of problem isolates by auxotyping, serotyping, and with a fluorescent antibody reagent

GonoGen (Micro-Media Systems, Potomac, MD) is a commercially available coagglutination test for the identification of Neisseria gonorrhoeae . We tested 84 strains of Neisseria spp. and Branhamella catarrhalis , including 50 clinical isolates of N. gonorrhoeae . Eighty-six percent (51/59) of N. gonorrhoeae , including 86% (43/50) of clinical isolates of N. gonorrhoeae , were identified correctly in the GonoGen test. In contrast, all N. gonorrhoeae isolates reacted with a fluorescent antibody research reagent composed of monoclonal antibodies, Both reagents were specific for N. gonorrhoeae . Clinical isolates were classified by auxotyping and were serotyped with research monoclonal antibody reagents in coagglutination tests to characterize problem isolates; two auxotype/serovar classes, prototrophic /IA-4 and proline-requiring/IA-4, accounted for 71% (5/7) of GonoGen-negative clinical isolates. Five of the seven isolates that were missed with the first GonoGen lot we tested did react with a second lot of GonoGen reagent. Investigators from different cities in the U.S. have reported different rates of success with GonoGen. Our results indicate that certain N. gonorrhoeae serovars may account for the difference in performance observed with serological tests.

Multiresistance plasmid from commensal Neisseria strains.

Antibiotic-resistant commensal strains of Neisseria spp. and Branhamella catarrhalis were isolated from throat cultures, on the basis of their capacity to grow in the presence of penicillin, streptomycin, or sulfamethoxazole-trimethoprim. Several strains, which belonged to different species of Neisseria, were resistant to beta-lactams, streptomycin, sulfamethoxazole, and trimethoprim, harbored a 6.0-megadalton plasmid with identical HinfI restriction patterns, and produced beta-lactamase and streptomycin phosphotransferase. The resistance determinants for beta-lactams, streptomycin, and sulfamethoxazole, but not for trimethoprim, were transferred from all these strains to Escherichia coli by conjugation or transformation. The resulting transconjugants or transformants acquired the plasmid and the capacity to produce beta-lactamase and streptomycin phosphotransferase. The 6.0-megadalton plasmid complemented a mutation which determines production of thermosensitive dihydropteroate synthetase in E. coli. We conclude that an R plasmid coding for beta-lactamase, streptomycin phosphotransferase, and a sulfonamide-resistant dihydropteroate synthetase is common to these strains.

Sulphur nutrition and metabolism in various species of Neisseria

Most Neisseria strains are able to grow with sulphate as a unique source of sulphur. Nevertheless, a cysteine requirement was present in a few strains of N. meningitidis and in 30% of N. flava strains isolated in our laboratory. All strains of N. gonorrhoeae exhibited such a requirement. In every strain tested, the need for cysteine (which can be satisfied by thiosulphate) was linked to the lack of sulphite-reducing-activity. The implications of these findings for the taxonomy and identification of Neisseria are discussed.

A new taxon in the genus Neisseria

Among 3,520 strains of Neisseria and Branhamella received in the laboratory during the last seven years, 13 could not be speciated. These 13 strains (Neisseria sp. group) were isolated from the throats of healthy children in Europe and Africa. The Neisseria sp. group oxidized glucose and maltose similarly to N. meningitidis. The observation of polysaccharide synthesis prompted the authors to examine the genomic relationship between the Neisseria sp. group and the type strains of all species of Neisseria and Branhamella. Additional phenotypic characteristics were also examined. Results indicated that the 13 strains in this study belong to the genus Neisseria, in which they constitute one homogenous hybridization group. They belong to the same genospecies as N. gonorrhoeae, N. meningitidis, N. flavescens and N. lactamica. However, they differ from N. meningitidis in production of polysaccharides, growth requirements and the lack of gamma-glutamyl transferase activity.