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.

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.

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.

Interrelationship of heat and relative humidity in the destruction of Clostridium botulinum type E spores on whitefish chubs.

Heat destruction of types B and E Clostridium botulinum spores on whitefish chubs was observed to be dependent upon the relative humidity (RH) in the chamber in which fish were heated. Experimental conditions were designed to simulate those attainable in commercial fish-smoking plants. Low numbers of type E spores were destroyed with regularity, within 30 min, on fish which were held at an internal temperature of 77 C (170.6 F) in an atmosphere of at least 70% RH. However, an internal temperature of 82 C (179.6 F) and a minimum RH of 70% were required to destroy several hundred thousand type E spores. Quantitative estimates of spore destruction were arrived at with a modified most probable number procedure. Type E spore populations were reduced by 2 to 4 logarithms at 77 C (170.6 F), by 5 to 6 logarithms at 82 C (179.6 F), and by more than 6 logarithms at 88 C (190.4 F) when fish were heated in an atmosphere of 70% RH. A 5 to 6 logarithm reduction of spores was also observed when fish inoculated with type B spores were processed at 82 C (179.6 F) in an atmosphere of 70% RH.

Sensitivity of an enrichment culture procedure for detection of Clostridium botulinum type E in raw and smoked whitefish chubs.

The sensitivity of an enrichment culture procedure for detecting Clostridium botulinum type E in whitefish chubs (Leucichthys sp.) was assayed. Data demonstrated that fish inoculated with 10 or more viable C. botulinum spores regularly develop specifically neutralizable enrichment cultures. Mild heat treatment (60 C, 15 min) substantially reduced the sensitivity of enrichment culturing. This effect was particularly noticeable in the culturing of fish which harbored fewer than 10 spores each. Evidence is presented which indicates that sensitivity of enrichment, without heat, approaches the level of one spore per fish. Smoked whitefish chubs, containing from one to several hundred spores each, were examined for toxin content after storage at 5, 10, 15, and 28 C for as long as 32 days. The lowest temperature at which detectable toxin was produced was 15 C. This occurred in 1 of 10 fish incubated for 14 days. C. botulinum was regularly recovered, by enrichment culture, from fish inoculated with small numbers of spores, even though toxin was not detected by direct extraction of incubated fish. Persistence of C. botulinum type E spores was observed to decline with an increase in the temperature and time at which inoculated fish were stored.

Demonstration and isolation of Clostridium botulinum types from whitefish chubs collected at fish smoking plants of the Milwaukee area.

A total of 1,071 whitefish chub samples were examined at eight stages of processing, including sampling aboard ship, various processing steps in the smoking plant, and display in retail cases. The frequency of Clostridium botulinum contamination of freshly caught and eviscerated chubs was approximately 13 to 14%. The highest percentage of contamination (20%) was noted among chubs sampled at the brining step of processing. The prevalence of contamination among chubs sampled at other processing stages prior to the smoking operation ranged from 6 to 14%. Of 858 freshly smoked chubs that had been processed at 180 F for 30 min (internal temperature of loin muscle), 10 were contaminated with C. botulinum (1 Type B and 9 Type E). The use of heat-shocked (60 C for 15 min) and nonheat-shocked enrichment cultures in combination yielded a greater number of positive samples than either method yielded when used alone. Each toxic enrichment culture obtained was subcultured to obtain isolation of the toxigenic organism. Toxigenic pure cultures of C. botulinum were obtained from 80% of the fish samples observed to produce toxic enrichment cultures.