Microbial film formation: dental plaque deposition on acrylic tiles using continuous culture techniques.

A chemostat system has been developed to model the attachment of oral bacteria, and the subsequent development of plaque film, to acrylic surfaces immersed in steady state cultures. Plaque was removed from the teeth and gingival margin of volunteers who refrained from oral hygiene for at least 72 h. Samples were pooled and inoculated into a complex growth medium maintained at 37 degrees C. Glucose-limited continuous culture was established at a dilution rate of 0.05/h and at pH 7.0. Microbiological analysis of the culture indicated that a complex community of oral bacteria was established, typical of that found in dental plaque. Acrylic tiles were immersed in the fermenter through a modified fermenter head and incubated therein for up to 21 d. Scanning electron microscopy showed that either side of the tiles contained a rough and a smooth surface and these initially favoured the attachment of fusiform bacteria, particularly on the rough surface. Cocci attached to those surfaces which were not heavily colonized by the fusiforms and eventually grew into and on the colonial sheets of the fusiforms.

Detection of streptococcal mutants presumed to be defective in sugar catabolism.

The tetrazolium method for detection of bacterial mutants defective in sugar catabolism was modified for use with streptococci. The critical factors were (i) the concentration of tetrazolium, which must be titrated to determine the optimum concentration for each species or even strain, and (ii) anaerobic incubation of tetrazolium-containing agar plates. When used with standard mutagenesis protocols, this method yielded lactose-negative mutants of nine streptococcal strains representing six species. A collection of lactose-negative mutants of streptococcus, sanguis Challis was characterized and contained phospho-beta-galactosidase, lactose phosphotransferase, and general phosphotransferase mutants.

Clustering of mutations affecting central pathway enzymes of carbohydrate catabolism in Pseudomonas aeruginosa.

Mutations in carbohydrate-negative mutants of Pseudomonas aeruginosa PAO1 individually deficient in glucose 6-phosphate dehydrogenase (zwf), 6-phosphogluconate dehydratase (edd), or pyruvate carboxylase (pyc) were mapped on the chromosome by plasmid R68.45-mediated conjugation and by bacteriophage F116L-mediated transduction. Loci for all three genes were located in the 45- to 55-min region of the chromosome; both zwf-1 and edd-1 were linked by transduction to nalA, whereas pyc-2 was linked by conjugation to argF10. The zwf-1 mutation exhibited cotransduction frequencies of greater than 95% with both edd-1 and the hex-9001 marker, a mutation reported to prevent growth on hexoses. The latter mutation was shown to cause a specific deficiency in 2-keto-3-deoxy-6-phosphogluconate aldolase activity and was redesignated eda-9001. These results demonstrate tight clustering of the gene loci for glucose 6-phosphate dehydrogenase and for both enzymes unique to the Entner-Doudoroff pathway in P. aeruginosa. Our evidence suggests supraoperonic clustering of these and other inducible carbohydrate catabolic genes in the 45- to 55-min region of the chromosome.

The threat of hepatitis B from dental school patients. A one-year study.

A study was conducted at the Louisiana State University School of Dentistry using the radioimmunoassay technique of serum analysis for presence of hepatitis B surface antigen (HBsAg) in all new patients accepted for treatment over a period of one year. The prevalence was 0.61 percent (22 seropositive patients from a total of 3,626 patients screened). Chronic HBsAg carriers, presence of e antigen, and high titers of antibody to hepatitis B core antigen (anti-HBc) were documented. Eighteen of the 22 seropositive patients had no past history of hepatitis, indicating the inadequacy of a history for revealing carriers of HBsAg.

Mannitol and fructose catabolic pathways of Pseudomonas aeruginosa carbohydrate-negative mutants and pleiotropic effects of certain enzyme deficiencies.

Mutant strains of Pseudomonas aeruginosa PAO were isolated on the basis of their inability to utilize mannitol as sole carbon source for growth. Four linkage groups (I through IV) among these mutant strains were resolved by two-factor crosses using the general transducing phage F116, and the strains appeared to contain point mutations as evidenced by ability to give rise to spontaneous revertants with wild phenotype on mannitol minimal agar. Group I strains were affected only in ability to grow on mannitol; all were deficient in inducible mannitol dehydrogenase activity, and all but one were deficient in inducible mannitol transport activity. Fructokinase was induced in group I strains and in wild-type bacteria during growth in the presence of mannitol but not fructose, indicating the presence of a pathway specific for endogenously generated fructose. Cells grown on fructose contained phosphoenolpyruvate:fructose-1-phosphotransferase activity, and mannitol-grown cells contained a lower level of this activity. Group II mutants were deficient in constitutive phosphoglucoisomerase, failed to grow on mannitol, grew very slowly on glycerol and fructose, but grew normally on glucose and gluconate. Group III strains were deficient in both nicotinamide adenine dinucleotide- and nicotinamide adenine dinucleotide phosphate-linked glucose-6-phosphate dehydrogenase activities that reside in a single enzyme species. 6-Phosphogluconate appeared to be the inductive effector for this enzyme, which was not required for aerobic growth on glucose or gluconate. A single mannitol-negative mutant in group IV also failed to grow on glycerol and glucose, but no biochemical lesion was identified.

Interspecies transformation of streptomycin resistance in oral streptococci.

Donor deoxyribonucleic acid extracted from streptomycin-resistant (Str(R)) mutant derivatives of a variety of strains of Streptococcus mutans, S. salivarius, and S. sanguis was used to transform streptomycin resistance into competent S. sanguis strain Challis. Transfer of genetic markers for sorbitol and mannitol fermentation and for extracellular polysaccharide as demonstrated by colonial morphology was not detected in this study. Reciprocal transformation between strain Challis and other oral streptococci could not be demonstrated. Transformation frequencies for Str(R) were relatively efficient among S. sanguis strains, with lower but significant frequencies demonstrated with strain Challis and donor deoxyribonucleic acid derived from other oral streptococci.

6-Phosphogluconate dehydratase deficiency in pleiotropic carbohydrate-negative mutant strains of Pseudomonas aeruginosa.

Mutants of Pseudomonas aeruginosa, strain PAO, have been isolated that are unable to grow on mannitol, glucose, gluconate, or 2-ketogluconate, cut that exhibit wild-type growth on pyruvate, lactate, citrate, succinate, or acetate. Although some of these mutants were also unable to grow on glycerol, the mutations formed a single linkage group by quantitative transductional analysis with phage F116 on glucose minimal agar medium. Cell extracts of all mutant strains were either lacking or severely deficient in 6-phosphogluconate dehydratase activity. Glu+ transductants derived from mutant strains that retained the wild-type ability for growth at the expense of glycerol also regained the ability to grow on all C-6 compounds. Although a role for the pentose phosphate pathway in the catabolism of C6 substrates was not found, a functional Entner-Doudoroff pathway appears to be essential for the catabolism of mannitol, glucose, gluconate, and 2-ketogluconate.

Pyruvate carboxylase deficiency in pleiotropic carbohydrate-negative mutant strains of Pseudomonas aeruginosa.

Cell extracts of Pseudomonas aeruginosa strain PAO were found to contain pyruvate carboxylase activity. Specific activities were minimal when cells were grown on Casamino Acids, acetate, or succinate, but were three- to fourfold higher when cells were grown in glucose, gluconate, glycerol, lactate, or pyruvate minimal media. The reaction in crude cell extracts and in partially purified preparations was dependent on pyruvate, adenosine 5'-triphosphate, and Mg(2+), but was not affected by either the presence or absence of acetyl coenzyme A. Activity was nearly totally inhibited by avidin and this inhibition was substantially blocked by free biotin in incubation mixtures. Cell extracts were shown to fix (14)CO(2) in a reaction that had these same characteristics. Eight pleiotropic, carbohydrate-negative mutant strains of the organism were isolated after nitrosoguanidine mutagenesis. Each mutant strain grew normally in acetate, succinate, and citrate minimal media but failed to utilize glucose, gluconate, 2-ketogluconate, mannitol, glycerol, lactate, and pyruvate as sole sources of carbon and energy. These strains were found by quantitative transductional analysis with phage F116 to form a single linkage group. Cell extracts of each mutant strain were either lacking or severely deficient in pyruvate carboxylase activity. Spontaneous revertants of five of the eight strains were isolated and found to recover simultaneously both pyruvate carboxylase activity and the ability to utilize each of the C(6) and C(3) compounds. A second linkage group of similar mutant strains that grew on the C(3) compounds was found to contain normal levels of pyruvate carboxylase activity, but each strain was deficient in an enzyme of the Entner-Doudoroff pathway.

Incidence of infectious drug resistance among fecal coliforms isolated from raw sewage.

Raw sewage was examined for the incidence of antibiotic-resistant coliforms present among both total and fecal coliforms. In both groups, it was found that approximately 3% of the coliform bacteria were resistant to two or more antibiotics. Of these organisms, 48% were capable of transferring all or part of their antibiotic resistance to an antibiotic-sensitive, F(-), derivative of Escherichia coli K-12. Among the R factors identified, those conferring resistance to streptomycin-tetracycline, ampicillin-streptomycin-tetracycline, and ampicillin or ampicillin-streptomycin accounted for 23, 20, and 15%, respectively, of the total R factors detected. The data indicate a significant level of infectious drug resistance among the fecal coliforms of the urban population. The data indicate further that because of the high incidence of coliform bacteria found to be doubly resistant to streptomycin and tetracyline, the inclusion of these antibiotics in selective media used for routine total or fecal coliform counts may serve to identify domestic sources of pollution.

Effect of antibiotic treatment on the incidence of infectious drug resistance among intestinal lactose-fermenting bacteria isolated from burn patients.

Antibiotic-resistant lactose-fermenting bacteria were recovered from the feces of 20 of 25 burn patients studied. Of the Escherichia coli isolated from patients receiving antibiotic treatment, 81.5% were shown to be infectiously resistant; only 32% of the E. coli recovered from patients not receiving antibiotics were shown to be harboring R factors. Three resistance patterns, ampicillin-chloramphenicol-streptomycin-kanamycin, ampicillin-tetracycline, and ampicillin-streptomycin, accounted for 25.2, 20.3, and 15.4%, respectively, of the total R factors identified.

Incidence of infectious drug resistance among lactose-fermenting bacteria isolated from raw and treated sewage.

Raw and treated sewage samples were examined for antibiotic-resistant, lactose-fermenting bacteria. Approximately 1% of the total lactose-fermenting bacteria were multiply resistant. Of these organisms, 50% were capable of transferring all or part of their resistance to a drug-sensitive recipient. Only 43% of those isolated on media containing a single antibiotic were capable of resistance transfer, whereas 57% of those recovered on multiple antibiotic plates transferred resistance. R factors conferring resistance to chloramphenicol, streptomycin, and tetracycline; streptomycin and tetracycline; and ampicillin, streptomycin, and tetracycline accounted for 22, 19, and 15%, respectively, of those identified. The data indicate a significant level of infectious drug resistance among the intestinal bacteria of the urban population.

Transductional analysis of Pseudomonas aeruginosa methionineless auxotrophs.

Seventy-one methionineless and cysteineless auxotrophs of Pseudomonas aeruginosa were placed into nine groups on the basis of their growth on methionine precursors and the cross-feeding response. Transduction experiments with bacteriophage F116 indicated the presence of four linkage groups among the methionineless mutants and at least three among the cysteineless mutants. These studies suggested that the biosynthesis of methionine in P. aeruginosa is similar to that described in other microorganisms, although none of the mutants lacking the ability to methylate homocysteine grew with vitamin B(12) or S-adenosylmethionine.

Infectious drug resistance among clinically isolated Escherichia coli.

Of 398 strains of clinically isolated Escherichia coli from three Birmingham, Alabama, hospitals, 38% were found to be resistant to one or more drugs tested. Fifty-seven per cent of the resistant strains transferred all or a part of their resistance pattern to sensitive cells during mixed cultivation. Of the 152 resistant strains, 29.1% were singly resistant, and 70.5% were resistant to more than one drug. Of the multiply resistant strains, 61% transferred all or a part of their pattern. Strains isolated from Veterans Hospital patients demonstrated higher percentages of resistance than strains isolated from Children's Hospital patients. An extremely low incidence of infective drug resistance was noted among E. coli isolated from the stools of healthy hospital employees.