Gardnerella vaginalis: characteristics, clinical considerations, and controversies.

The clinical significance, Gram stain reaction, and genus affiliation of Gardnerella vaginalis have been controversial since Gardner and Dukes described the organism as the cause of "nonspecific vaginitis," a common disease of women which is now called bacterial vaginosis. The organism was named G. vaginalis when taxonomic studies showed that it was unrelated to bacteria in various genera including Haemophilus and Corynebacterium. Electron microscopy and chemical analyses have elucidated the organism's gram-variable reaction. Controversy over the etiology of bacterial vaginosis was largely resolved by (i) studies using improved media and methods for the isolation and identification of bacteria in vaginal fluids and (ii) standardization of criteria for clinical and laboratory diagnosis. Besides G. vaginalis, Mobiluncus spp., Mycoplasma hominis, and certain obligate anaerobes are now acknowledged as participants in bacterial vaginosis. The finding that G. vaginalis, Mobiluncus spp., and M. hominis inhabit the rectum indicates a potential source of autoinfection in addition to sexual transmission. Extravaginal infections with G. vaginalis are increasingly recognized, especially when the toxic anticoagulant polyanetholesulfonate is omitted from blood cultures and when urine cultures are incubated anaerobically for 48 h. The finding that mares harbor G. vaginalis suggests that an equine model can be developed for studies of Gardnerella pathogenesis.

Branhamella catarrhalis: an organism gaining respect as a pathogen.

Branhamella catarrhalis was formerly regarded as a common, essentially harmless inhabitant of the pharynx. This misapprehension was caused, in part, by confusion with another pharyngeal resident, Neisseria cinerea. The two organisms can now be differentiated by the positive reactions of B. catarrhalis in tests for nitrate reduction and hydrolysis of tributyrin and DNase. B. catarrhalis is currently recognized as the third most frequent cause of acute otitis media and acute sinusitis in young children. It often causes acute exacerbations of chronic bronchopulmonary disease in older or immunocompromised adults and is incriminated occasionally in meningitis, endocarditis, bacteremia, conjunctivitis, keratitis, and urogenital infections. Virulence-associated factors, such as pili, capsules, outer membrane vesicles, iron acquisition proteins, histamine-synthesizing ability, resistance to the bactericidal action of normal human serum, and binding to the C1q complement component, have been identified in some strains. beta-Lactamase producing strains, first detected in 1976, have risen to approximately 75% worldwide. Thus far, however, practically all American strains of B. catarrhalis remain susceptible to alternative antibiotics. A possible selective advantage of recent isolates is their reportedly heightened tendency for adherence to oropharyngeal cells from patients with chronic bronchopulmonary disease.

Genetic basis of the association of sulphonamide resistance with methionine auxotrophy in Neisseria gonorrhoeae.

Selection by sulphonamides was investigated in Neisseria gonorrhoeae because a sulphonamide-resistant (Sulr), methionine-requiring (Met-) phenotype that was common in the era of sulphonamide therapy became rare in the penicillin era. Cultures of wild-type (SulsMet+) gonococci on a conventional medium containing sulphadiazine (2-10 micrograms ml-1) yielded numerous, nonidentical mutations of two met genes. The requirement of MetI- mutants was satisfied only by methionine, whereas MetII- mutants utilized either homocysteine or methionine. My theory that increased resistance to sulphonamides is a pleiotropic effect of methionine auxotrophy was confirmed by the return of sulphonamide susceptibility in all Met+ spontaneous mutants. Furthermore, the SulrMet- traits were introduced or eliminated together by DNA-mediated transformation. Sulphonamides are known to inhibit dihydropteroate synthase; consequently, they interrupt the entire sequence of reactions in the folate pathway including the methyl group transfer from N5-methyltetrahydrofolate to homocysteine to form methionine. The increased sulphonamide resistance of these Met- mutants is discussed in terms of conservation of the pool of essential tetrahydrofolate derivatives. The ease with which spontaneous forward and reverse met mutations can be obtained is unique among gonococcal genes.

Neisseria gonorrhoeae recombinant strains expressing hybrid serological reactivities of outer membrane proteins IA and IB.

The inheritance of epitopes of protein I, the principal protein of the outer membrane of Neisseria gonorrhoeae, was investigated by DNA-mediated transformation. Protein I transformants were isolated by selection for the linked spectinomycin-resistance determinant. Twelve monoclonal antibodies used in coagglutination tests identified epitopes of the two forms of protein I (P.IA and P.IB). A given gonococcal culture from patients expresses epitopes of either P.IA or P.IB and rarely, if ever, exhibits hybrid P.IA/P.IB reactivities. Nevertheless, we found 35 P.IA/P.IB recombinants among 1506 transformants. Transmission by DNA of the hybrid reactivities and the apparent molecular mass characteristic of a given P.IA/P.IB species verified the genetic basis of the phenotypic changes. A recombinant that expressed six P.IA and two P.IB epitopes is of interest as a possible component of a gonococcal vaccine, because one or more of these epitopes are shared with 99.8% of a worldwide collection of 1858 clinical strains.

Mutation in a locus linked to penB-nmp causes suppression of the Mtr phenotype of Neisseria gonorrhoeae.

The chromosomal locus mtr, which encodes low-level resistance to multiple antibacterial agents in Neisseria gonorrhoeae, is subject to phenotypic suppression by env mutations that increase the permeability of the envelope. We have identified a new locus, mom (for modifier of Mtr), which is located on the chromosome very close to penB and nmp, loci known to be linked to each other and to spc. Phenotypic suppression of Mtr was recognized by reductions of resistance to benzylpenicillin and also to oxacillin and the hydrophobic agents novobiocin and erythromycin. The resistance to each of these antibiotics returned to the Mtr levels in mom+ transformants isolated by selection for increased resistance to either novobiocin or erythromycin; the accompanying change of the outer membrane protein I seroreactions confirmed the proximity of nmp and mom. Thus, some mutant gonococci display wild-type antibiotic susceptibilities but can express multiple resistance following a mom+ mutation that releases the suppressed Mtr phenotype.

Vancomycin hypersusceptibility in Neisseria gonorrhoeae isolated from patients involves diverse mutations.

We investigated the genetic determinants of hypersusceptibility to vancomycin and erythromycin found in Neisseria gonorrhoeae strains isolated from patients. In terms of resistance (highest concentration of antibiotic permitting growth), the levels of vancomycin resistance of six strains ranged from 0.2 to 1.0 microgram/ml, and the level of erythromycin resistance of these strains was 0.02 or 0.05 micrograms/ml. DNA from these strains was used to introduce their hypersusceptibility determinants into partially isogenic derivatives of N. gonorrhoeae 89 which initially had wild-type levels of resistance to vancomycin (greater than or equal to 3.0 micrograms/ml) and erythromycin (greater than or equal to 0.1 microgram/ml). The recombination frequencies found in reciprocal transformation tests of six isogenic strains indicated that the mutations responsible for vancomycin hypersusceptibility were located at different sites. The transformants selected for increased resistance to vancomycin were also resistant to erythromycin. This evidence, together with DNA concentration-response curves, indicated that the mutations affected either one gene locus or closely linked loci. The recombination indices obtained in crosses between our hypersusceptible strains and DNAs from reference strains carrying the envelope mutations env-1, env-2, env-3, and env-10 showed that the mutation (designated env-12) responsible for erythromycin hypersusceptibility in one strain (89-954) was located in close proximity to env-2. The determinant of vancomycin hypersusceptibility in strain 89-954 was distinct from env-12, but the two were linked. In the other five isogenic strains, the hypersusceptibilities to both vancomycin and erythromycin could be annulled by spontaneous mutations in a locus provisionally designated vel because of its likely effects on the envelope. Vel+ mutants obtained by selection with either vancomycin alone or erythromycin alone gained increased resistance to both antibiotics.

Characteristics of Neisseria gonorrhoeae strains isolated on selective and nonselective media.

The possible inhibition of Neisseria gonorrhoeae on modified Thayer-Martin (VCNT) medium was investigated by inoculation of multiple media with specimens from 3,490 patients. N. gonorrhoeae was recovered from 461 patients, and in 24 cases (5.2%) it was isolated on drug-free medium only; 18% of the recoveries were on VCNT medium only. The minimal inhibitory concentration (MIC) of benzylpenicillin was determined for 411 of the strains, and 175 were examined for responses to 12 other antibiotics and for auxotype. Of the 24 strains isolated on drug-free medium only, one was inhibited by trimethoprim at a concentration of 2.0 micrograms/ml and four others had MICs of vancomycin of less than or equal to 2.0 micrograms/ml. The remainder were resistant to vancomycin, trimethoprim, and colistin at the concentrations present in VCNT medium. Unexpectedly, four strains isolated on both VCNT and drug-free medium had MICs of vancomycin of less than or equal to 3.0 micrograms/ml and were defined as hypersusceptible. Genetic tests showed that gonococci resistant to less than or equal to 0.5 microgram of vancomycin/ml differed genotypically from those resistant to 1.0 microgram/ml. The eight strains hypersusceptible to vancomycin were highly susceptible to various other antibiotics. Their nutritional requirements included hypoxanthine (Hyx-) and uracil (Ura-), and all but one also required arginine (Arg-), which for two strains could not be replaced by ornithine (Arg0-). Pro-,Arg0-,Ura- (5.7%) and Arg0-,Ura- (1.1%) auxotypes were found at this time but not in earlier studies of gonococci isolated in the same clinic.

Probenecid: antibacterial action against Neisseria gonorrhoeae and interaction with benzylpenicillin.

Neisseria gonorrhoeae strains were used in an investigation of the antibacterial action of probenecid and its interaction with benzylpenicillin. The growth of 112 routine isolates was inhibited by probenecid at concentrations of 100 to 500 micrograms/ml incorporated in agar. Additive or synergistic effects of benzylpenicillin-probenecid combinations were graphically illustrated in gradient plates. In agar dilution tests with a resistant gonococcal strain, the MICs of benzylpenicillin alone and probenecid alone were 0.8 and 500 micrograms/ml, respectively; in contrast, the MICs of combinations of benzylpenicillin and probenecid were 0.45 and 75 micrograms/ml and 0.3 and 150 micrograms/ml, respectively. High concentrations of probenecid in broth were bactericidal. Probenecid alone at 50 to 100 micrograms/ml had little antibacterial effect, but in combination with an appropriate concentration of benzylpenicillin, it produced reductions of CFU in 6 h that were 100 to 300 times that produced by benzylpenicillin alone. Thus, in addition to its well-known pharmacological effects, probenecid potentiates the in vitro action of benzylpenicillin for gonococci. I suggest that synergism contributes to the beneficial effect of the benzylpenicillin-probenecid regimen for the treatment of gonorrhea. Furthermore, synergism may explain the reduction in the ratio of partially benzylpenicillin-resistant N. gonorrhoeae strains to benzylpenicillin-susceptible strains that occurred in the United States between 1972 and 1978.

Arginine and pyrimidine biosynthetic defects in Neisseria gonorrhoeae strains isolated from patients.

Neisseria gonorrhoeae strains with nutritional requirements that include arginine (Arg-), uracil (Ura-), and hypoxanthine have attracted attention because of their tendency to cause disseminated infections, as a basis for genetic studies of arginine and pyrimidine biosynthesis, we examined the activities of four enzymes of these pathways in cell-free extracts of both prototrophic and Arg- Ura- strains. Activities of glutamate acetyltransferase, aspartate transcarbamylase, and orotate phosphoribosyltransferase, encoded respectively by argE, pyrB, and pyrE, were absent in some Arg- Ura- isolates. Gonococci that were unable to utilize ornithine for growth in place of citrulline lacked activity of carbamyl phosphate synthetase (encoded by car). Defects of car imposed requirements for both citrulline (or arginine) and a pyrimidine because of the dual role of carbamyl phosphate in the two pathways. Defects of argE, car, pyrB, and pyrE were separately introduced by genetic transformation into representatives of a gonococcal strain which initially was prototrophic. Results of enzyme assays of these isogenic auxotrophic transformants confirmed the gene-enzyme relationships.

Neisseria gonorrhoeae isolated from disseminated and localised infections in pre-penicillin era. Auxotypes and antibacterial drug resistances.

Interest in the evolution of gonococcal auxotrophy led to a study of 72 strains isolated between 1935 and 1948 from the urogenital tract (57 patients), the eye (two patients), and from disseminated gonococcal infections (11 patients and probably two others). Two cervical isolates with nutritional requirements for proline, arginine, histidine, and biotin were oxidase-positive, Gram-negative diplococci, but their identity as Neisseria gonorrhoeae was uncertain because they were atypically susceptible to colistin and did not produce acid in glucose media. The N gonorrhoeae strains were highly susceptible to 11 other antibacterial drugs but not to sulphadiazine. Defects of one or more pathways for the biosynthesis of methionine, proline, arginine, threonine, lysine, the branched-chain amino acids, hypoxanthine, and thiamine pyrophosphate were found in 39 of the 70 strains, including four isolated in the presulphanilamide era. Unexpectedly, methionine was required for the growth of 11 (21%) of the 52 Danish strains and for 13 (72%) of 18 strains isolated in the USA. The Danish strains included 28 (54%) that did not require any of the compounds used for differentiating auxotypes, whereas this type was represented by only three (17%) of the USA strains. None of the gonococci required uracil or other pyrimidines. This suggests that the requirements for arginine, hypoxanthine, and uracil commonly found in recent isolates from disseminated gonococcal infections probably evolved treatment with sulphonamide was replaced by penicillin.

Auxotypes and antibacterial resistance to gonococci with differing susceptibilities to vancomycin.

The responses to vancomycin and 11 other antibacterial drugs and the nutritional requirements of gonococci recovered from two selective media were determined. Single urogenital specimens from 508 patients attending a social hygiene clinic in 1975 yielded 97 strains of Neisseria gonorrhoeae; 95 were recovered on VCNT (a modification of Thayer-Martin medium), always inoculated first, and 69 on LC medium containing lincomycin (4 micrograms/ml) and colistin (5 micrograms/ml). The two drugs at these concentrations in LC medium were not inhibitory for isolates from either medium. Unexpectedly, three isolates on VCNT were susceptible to vancomycin at the concentrations (3 micrograms/ml) in VCNT medium; these three were typically sensitive to penicillins but were hypersusceptible to erythromycin (inhibited by less than or greater than 0.05 micrograms/ml) and rifampin (less than or equal to 0.02 micrograms/ml). Resistance to streptomycin (greater than or equal to 500 micrograms/ml) (22% of the strains) was correlated with increased resistance to penicillins, erythromycin, and rifampin in most instances. All streptomycin-resistant gonococci required proline, or arginine, or none of the test compounds. Strains requiring arginine, hypoxanthine, and uracil were uniformly sensitive to antibiotics but not hypersusceptible. In contrast, six strains of N gonorrhoeae isolated in Denmark required arginine (not satisfied by ornithine), hypoxanthine, and uracil and were hypersusceptible to vancomycin (inhibited by 0.5 micrograms/ml), erythromycin, and rifampin. DNA-mediated transformation showed that all three hypersusceptibilities of one Danish strain were introduced together into a wild-type gonococcus, suggesting that a mutation of an env (envelope) locus might be responsible for the atypical permeability.

Arginine biosynthesis in Neisseria gonorrhoeae: enzymes catalyzing the formation of ornithine and citrulline.

Many of the Neisseria gonorrhoeae strains isolated from patients require arginine for growth in a defined medium. As a basis for genetic studies of these Arg- strains, we examined two biosynthetic enzymes of Arg+ (nonrequiring) gonococci. Cell-free extracts contained (i) glutamate acetyltransferase, which catalyzes the formation of L-ornithine from alpha-N-acetyl-L-ornithine, and (ii) ornithine transcaramylase, which catalyzes the reaction between L-ornithine and carbamyl phosphate, yielding L-citrulline. Arg- strains were unable to utilze alpha-N-acetyl-L-ornithine for growth lacked significant activity of glutamate acetyltransferase, and activity was gained by Arg+ clones derived by DNA-mediated transformation. Some of the Arg- patient isolates were unable to use either alpha-N-acetyl-L-ornithine or L-ornithine in place of arginine, and two separate steps of genetic transformation were required to yield Arg+ cells. Extracts of these doubly auxotrophic cells lacked glutamate acetyltransferase activity, but, unexpectedly, they displayed normal ornithine transcarbamylase activity. This finding illustrates the importance of identifying the products specified by arg loci during genetic studies of arginine auxotrophy.

Nutritional requirements and penicillin susceptibilities of gonococci from pharyngeal and anogenital sites.

Neisseria gonorrhoeae was isolated from 5.9% of oropharyngeal specimens obtained from patients attending a clinic for sexually transmitted diseases. Oropharyngeal isolates from 69 patients and anogenital isolated from 97 other patients attending the same clinic were compared. Many of the gonococci could be differentiated by the compounds required for growth in chemically defined media or by differences in the minimum inhibitory concentration (MIC) of penicillin G. Strains with requirements for either proline (Pro-) or arginine (Arg-) or for none of the compounds that are used for differentiation (zero phenotype) were more common in the oropharynx (91.3% of patients) than in anogenital sites (73.2% of patients). On the other hand, gonococci with multiple requirements that include arginine, hypoxanthine, and uracil (AHU strains) were present in oropharyngeal specimens from only three patients (4.4%), but were isolated from anogenital specimens from 18 patients (18.6%). A high susceptibility to penicillin characterised the AHU strains from all sites, as others have reported. The penicillin MIC ranged from 0.003-0.72 microgram/ml for strains with Pro-, Arg-, and zero phenotypes. However, a penicillin MIC greater than or equal to 0.42 microgram/ml was found for 17.6% of oropharyngeal isolates of these types, but for only 4.1% of Pro-, Arg-, and zero isolates from anogenital sites. None of these moderately resistant strains produced beta-lactamase. Our findings indicate that gonococci differ in their ability to colonise the oropharynx successfully.

Auxotypes and penicillin susceptibilities of Neisseria gonorrhoeae isolated from patients with gonorrhea involving two or more sites.

A system of auxotyping described in 1973 is based on the differing nutritional requirement patterns of Neisseria gonorrhoeae strains. Our ongoing evaluation of the reliability of auxotyping has involved a study of the constancy of characteristics of gonococci isolated at one time from two or more sites of a given subject. The auxotypes and minimal inhibitory concentration (MIC) of penicillin G were determined for 181 isolates obtained from 84 patients with uncomplicated gonorrhea, for 16 isolates from 8 couples with uncomplicated gonorrhea, and for 21 isolates from 12 other patients, 9 with disseminated gonococcal infection and three consorts. The penicillin MIC served to distinguish between many members of auxotypes 1, 2, and 3, which are commonly involved in uncomplicated gonorrhea. Thus, for proline-requiring gonococci (auxotype 2) the MIC ranged from 0.01 to 1.2 IU of penicillin per ml. The profile of gonococcal responses to seven other antibacterial drugs provided useful additional information where the extent of phenotypic similarity was in doubt. In all but seven instances, the gonococci isolated from different sites of the same patient, or from a consort, had the same nutritional requirements and penicillin MIC. The gonococci isolated from one patient with disseminated gonococcal infection and from one of her two sexual contacts had nutritional requirements for arginine, hypoxanthine, uracil, and thiamine pyrophosphate, whereas the strain isolated from her second contact differed in having no requirement for thiamine pyrophosphate. The paired cervical and rectal isolates from one patient with uncomplicated gonorrhea differed only with respect to a requirement for hypoxanthine. Pairs of isolates from three patients differed slightly in degree of susceptibility to penicillin. In the remaining two instances, however, numerous differences between the isolates from the endocervix and the anal canal of a given patient indicated the presence of concomitant infections with different strains of N. gonorrhoeae.

[Biochemical differentiation between N. sicca and N. perflava (author's transl)]. / Biochemische Unterscheidung von Neisseria sicca und Neisseria perflava

Neisseria sicca forms an iodine-positive metabolic product when grown on Trypticase soy agar without the addition of sucrose. This reaction which does not occur with N. perflava may be useful for differentiating between the two species.

Iodometric detection of Haemophilus influenzae beta-lactamase: rapid presumptive test for ampicillin resistance.

Strains of Haemophilus influenzae type b sporadically isolated from clinical specimens are ampicillin resistant due to production of a beta-lactamase. This enzyme which inactivates ampicillin and penicillin G is not produced by ampicillin-susceptible strains. Various characteristics of beta-lactamase production and ampicillin resistance of three H. influenzae type b isolates were investigated. A sensitive iodometric test was employed to detect beta-lactamase; positive results were obtained in 5 min with 10(9) bacteria taken from cultures on a nutritionally adequate agar medium. This simple chemical test will enable the hospital laboratory to obtain presumptive evidence of ampicillin resistance on the same day that H. influenzae is isolated.

Cellular elongation under the influence of antibacterial agents: way to differentiate coccobacilli from cocci.

Representatives of Moraxella, Acinetobacter, and various other groups of short, gram-negative bacilli are readily distinguished from Neisseria by microscopic observation of filaments produced by the rods during growth in the presence of low concentrations of penicillins or sulfadiazine. Wet mounts of bacteria from routine antibiotic susceptibility test cultures are satisfactory for examination of morphology.

Genetic transformation of biosynthetically defective Neisseria gonorrhoeae clinical isolates.

Deoxyribonucleic acid (DNA) and chemically defined media were used in transformation tests of 51 strains of Neisseria gonorrhoeae which exhibited various biosynthetic defects when isolated from patients. These auxotrophic gonococci had one or more nutritional requirements involving proline, methionine, arginine, hypoxanthine, uracil, and thiamine pyrophosphate (THPP). DNA from a clinical isolate which did not require these compounds for growth on defined medium transformed each of the auxotrophic markers of all 51 recipient populations. Ten isolates had defects involving the synthesis of THPP; four strains (designated Thp(-)) had a growth requirement that was satisfied only by THPP, whereas the requirement of six strains (designated Thi(-)) was satisfied by either thiamine or THPP. DNA from Thp(-) donors elicited transformation of Thp(-) as well as Thi(-) recipients. Reciprocally, DNA from a Thi(-) donor transformed both Thi(-) and Thp(-) recipients. Furthermore, DNA from other auxotrophic gonococci had transforming activity for some phenotypically similar auxotrophic recipients. The findings indicate the existence of various nonidentical genetic defects which block reactions in the biosynthesis of proline, methionine, arginine, hypoxanthine, and THPP. Routine cultures from patients with gonorrhea were the source of these auxotrophic strains of N. gonorrhoeae; the various nutritional requirements were identified by a recently described system of gonococcal auxotyping. The transformation test results verify the hereditary basis of the auxotypes, establish that many different mutations exist in potentially virulent gonococci, and illustrate the value of these auxotrophic mutants for studies of the genetic structure and evolution of natural populations of gonococci.

Neisseria gonorrhoeae auxotyping: differentiation of clinical isolates based on growth responses on chemically defined media.

A system is described for differentiating clinïcal isolates of Neisseria gonorrhoeae based on their growth or absence of growth on a set of 11 chemically defined agar media. The complete medium, NEDA, contains all of the compounds required for gonococcal growth; but isolates differ in their ability to grow on NEDA from which selected compounds are individually omitted. The differential compounds include L-proline, L-arginine, L-ornithine, L-methionine, hypoxanthine, uracil, thiamine, and thiamine pyrophosphate. A distinctive pattern of growth responses on the standard media defines an auxotype. Twenty auxotypes were found among a group of 251 gonococci which were isolated from patients examined in the clinics of one city during a 3-month span of time. Another collection of 74 strains from several different countries yielded two additional auxotypes. The stability of the nutritional requirements on which the auxotyping depends was verified in two ways. Cultures isolated from different anatomic sites of a patient or from sexual partners represented the same auxotype, as did cultures which were repeatedly isolated from cases of presumptive treatment failures. Also, the auxotypes of gonococci remained the same after numerous subcultures. The reproducibility of results and the variety and number of auxotypes indicate the potential value of the auxotyping system as an epidemiological tool.