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.

Identification of Moraxella bovis by qualitative genetic transformation and nutritional assays.

Strains of Moraxella bovis were identified definitively through the combined use of a qualitative genetic transformation assay and determination of the ability of the organism under examination to grow in a defined medium (medium MB). Except for weak transformation by DNA from strains of M. lacunata, M. nonliquefaciens, and M. (Branhamella) ovis, DNA samples from all other members of the genus Moraxella failed to transform either of the two M. bovis auxotrophs used in this study.

Combined genetic transformation and nutritional assay for identification of Moraxella nonliquefaciens.

A combined genetic transformation and nutritional assay is described that permits definitive identification of clinically isolated strains of Moraxella nonliquefaciens. Crude DNA preparations of strains of various Moraxella species were used to transform nutritional mutants of a stably competent strain of M. nonliquefaciens for ability to grow on a defined medium (Mn-B). DNA samples from 24 independently isolated strains of M. nonliquefaciens all resulted in massive (4+) transformation of each of two mutant assay strains. DNA samples from strains of M. bovis and M. lacunata frequently gave weak (1+) transformation of one of the mutant assay strains (Mn64) but almost always failed to transform another assay strain (Mn136). DNA samples from eight other Moraxella species failed completely to transform either of the mutant assay strains. When streaked on the defined medium used for the transformation assay (Mn-B), 23 of the 24 strains of M. nonliquefaciens grew well, but all strains of M. bovis and M. lacunata failed to grow on this medium.

Defined medium for Moraxella bovis.

A defined medium (medium MB) for Moraxella bovis was formulated. Nineteen strains grew well on medium MB. One strain was auxotrophic for asparagine, and another was auxotrophic for methionine. Strains of M. equi and M. lacunata also grew on medium MB. All strains had an absolute requirement for thiamine and were stimulated by or actually required the other growth factors in the medium.

Defined medium for Moraxella (Branhamella) catarrhalis.

A defined medium for growth of 24 strains of Moraxella (Branhamella) catarrhalis was devised. This medium (medium B4) contains sodium lactate as a partial carbon source, proline as both a partial carbon source and a partial nitrogen source, aspartate as a partial nitrogen source, and the growth factors arginine, glycine, and methionine. Either aspartate, glutamate, or proline could serve as sole nitrogen source, but growth occurred at a significantly better rate if proline was present together with either aspartate or glutamate, or with both aspartate and glutamate. With the exception of strain ATCC 23246, all the strains had an absolute requirement for arginine and either a partial or absolute requirement for glycine. The concentration of glycine required for optimal growth was found to be relatively high for an amino acid growth factor. Heart infusion broth was found to be growth inhibitory for spontaneous mutants of one strain able to grow in the absence of arginine, and such mutants reverted readily to arginine dependence accompanied by the ability to grow faster on the complex medium. Growth rates in the defined medium B4 were enhanced by the simultaneous addition of asparagine, glutamate, glutamine, leucine, lysine, histidine, and phenylalanine.

General approach to bacterial nutrition: growth factor requirements of Moraxella nonliquefaciens.

A general procedure was devised for the determination of growth factor requirements of heterotrophic bacteria based upon identification of individual nutrients as they are successively depleted from a limited quantity of complex medium. By using this approach, it was possible to develop a defined medium for growth of Moraxella nonliquefaciens that contained nine amino acids and three vitamins. Three of the amino acids, proline, serine, and cysteine, were required in unusually high concentrations to obtain optimal growth. Methionine had a sparing action on the requirements for serine and cysteine. Glycine could substitute for serine. Although a required nutrient, cysteine was inhibitory for growth, but this inhibitory action was antagonized by valine or leucine. The requirement for cysteine was satisfied by cystine, glutathione, or sodium sulfide. M. nonliquefaciens could not use ammonia as a nitrogen source but could use glutamate or aspartate for this purpose. With the exception of 1 auxotrophic strain, the growth factor requirements of 23 independently isolated strains of M. nonliquefaciens were essentially the same.

Endophthalmitis caused by Moraxella nonliquefaciens.

We report the first case of endophthalmitis caused by Moraxella nonliquefaciens. Most likely, entry followed minor trauma from a contact lens, with the marked invasiveness of the organism resulting from the patient's immunocompromised state. This case, in addition, serves to illustrate the advantage of genetic transformation assay to differentiate the species of this group of organisms.

Transformation assay for identification of psychrotrophic achromobacters.

The finding that many psychrotrophic, gram-negative, nonmotile, oxidase-positive coccobacilli (achromobacters) are competent for genetic transformation made possible the development of a transformation assay that permits recognition of genetically related strains. It has been demonstrated that 109 independently isolated achromobacters are genetically related since deoxyribonucleic acid samples from all of these organisms were able to transform a single competent auxotrophic strain to prototrophy. Genetically interacting bacteria included strains that lacked one or more of the characteristics typical for most achromobacters. An oxidase-negative mutant of one of these strains reacted positively in the transformation assay, unlike other oxidase-negative bacteria. Achromobacters were derived from fish, poultry, irradiated foods, seawater, and other sources. One strain previously classified as Micrococcus cryophilus has been shown to be related to the achromobacters. Two achromobacters had an optimum growth temperature of 35 degrees C and behaved as typical mesophiles. The moraxellae and Acinetobacter were shown to be unrelated to the achromobacters by using the transformation assay. The ready demonstration of genetic relatedness provides a new basis for taxonomic grouping of the psychrotrophic achromobacters.

Studies of some naturally occurring auxotrophs of Neisseria gonorrhoeae.

A strain of Neisseria gonorrhoeae requiring arginine, proline, glutamate and cystine as nutritional supplements was transformed, in several steps, to grow in a simple mineral medium containing cystine as the only growth factor with DNA from several clinically isolated strains of this organism. Using DNA from naturally occurring auxotrophs (auxotypes) known to require arginine, hypoxanthine and uracil (Arg-Hyx-Ura-), as well as other factors, it was possible to transfer nutritional markers, one at a time, into such prototrophs to obtain seven single marker auxotrophic strains. Three different uracil markers, two different hypoxanthine markers, an arginine marker, and an isoleucine--valine markers were each introduced into separate strains. Of 114 DNA samples from independently isolated strains of N. gonorrhoeae, 54 were able to transform all seven single marker strains to prototrophy. Six of the single marker strains failed to be transformed to prototrophy by DNA samples from 43 strains, thus demonstrating that all these strains possess at least six nutritional lesions in common. Two strains were shown to contain all seven nutritional lesions, whereas several strains contained some but not all of the seven lesions. Six of the seven single marker strains have been shown to revert spontaneously to prototrophy at low frequencies. During construction of prototrophic strains it was observed that genes conferring sensitivity to growth inhibition by nutrients in complex media were occasionally transferred along with prototrophy.

Properties of mutants of Escherichia coli lacking malic dehydrogenase and their revertants.

Mutants of Escherichia coli lacking malic dehydrogenase activity (mdh) were incapable of growth on acetate", succinate- or malate/mineral medium. Revertants of mdh strains which had regained the ability to grow on C4-dicarboxylic acids could be divided into two distinct classes. One type of revertant had regained the ability to synthesize functional malic dehydrogenase. The other type of revertant still lacked malic dehydrogenase activity but possessed a suppressor mutation which altered the regulation of the synthesis or activity of the C4-dicarboxylic acid transport system, resulting in increased C4-dicarboxylic acid transport activity. This latter class of revertants apparently synthesized oxalacetate from malate via the sequential actions of the NAD-linked malic enzyme, phosphoenolpyruvate synthetase, and phosphoenolpyruvate carboxylase. Evidence has been presented that is consistent with the hypothesis that oxalacetate is the inducer of the C4-dicarboxylic acid transport system. The inability of mutants lacking malic dehydrogenase to grow with a C4-dicarboxylic acid as the carbon source can be attributed to the difficulty such mutants have in synthesizing oxalacetate.

Utilization of oxalacetate by Acinetobacter calcoaceticus: evidence for coupling between malic enzyme and malic dehydrogenase.

Growth of Acinetobacter calcoaceticus strain BD413 in malate-mineral medium resulted in the excretion of large quantities of oxalacetate. Malate was virtually depleted by the time the cell density reached 60% of its final value; most of the remaining growth took place at the expense of oxalacetate. Experiments in which oxalacetate was used as the initial substrate showed that pyruvate was not utilized until most of the oxalacetate disappeared. The generation time for growth on malate or oxalacetate was approximately 40 min; the generation time for growth on pyruvate was 62 min, which implies that pyruvate transport may be rate limiting. Oxalacetate and pyruvate, however, supported approximately the same growth yield. These observations suggested that the first step in the utilization of oxalacetate as an energy source consisted of an enzymatic decarboxylation of the keto acid to pyruvate and CO(2). Three enzyme reactions that carry out this decarboxylation have been detected in extracts of A. calcoaceticus. The first, which functioned maximally at pH 4.8, was attributable to the oxalacetate decarboxylase activity of oxidized diphosphopyridine nucleotide-malic enzyme. The second and third, which functioned in the neutral pH range, resulted from coupling of oxidized diphosphopyridine nucleotide-malic enzyme to reduced diphosphopyridine nucleotide-dependent malic dehydrogenase, and oxidized triphosphopyridine nucleotide-malic enzyme to a reduced triphosphopyridine nucleotide-dependent malic dehydrogenase. The efficiency of these coupled reactions was high enough so that the overall reaction could be physiologically significant.

Unusual effects of penicillin G and chloramphenicol on the growth of Moraxella osloensis.

Growth of exponential-phase liquid cultures of Moraxella osloensis was inhibited by 0.5 U of penicillin G per ml. For this organism, low concentrations of penicillin acted primarily in a bacteriostatic rather than in a bactericidal manner. At higher concentrations of penicillin some killing did take place, but the rate of killing was rather slow and appeared to be independent of penicillin concentration. Microscopic observation of cells from penicillin-treated cultures showed little or no cellular swelling or lysis. The total cell count did not decrease significantly during 6 h of incubation in 5,000 U of penicillin per ml. The rates of respiration, nucleic acid synthesis, and protein synthesis were not affected by the presence of penicillin. Attempts to counteract the bactericidal action of high concentrations of penicillin with growth inhibitory concentrations of chloramphenicol were unsuccessful, since chloramphenicol itself was more bactericidal than penicillin for M. osloensis.

Simple method for distinguishing gonococcal colony types.

Gonococcal colony types can be distinguished by a new procedure that makes use of a dissecting microscope with a concave mirror and a fluorescent lamp. Critical adjustment of the mirror angle results in illumination similar to that obtained in the dark-field microscope. When the concave mirror is set at a certain angle, colonies of the lenticular types 1 and 2 refract the light coming through them in such a way that an edge of the microscope stage is focused in each colony. By contrast, colonies of types 3 and 4, which are relatively flat, fail to refract incident light. Although distinguishable from each other by differences in color, type 3 and 4 colonies do not display the focusing effect typical for type 1 and 2 colonies and appear uniformly illuminated. This new technique permits the rapid identification and isolation of even a single type 1 or 2 colony in a field of type 3 or 4 colonies, making it possible to obtain and maintain competent colonies (type 1 or 2) for the genetic transformation assay for Neisseria gonorrhoeae strain identification as well as for other purposes.

Genetic transformation assays for identification of strains of Moraxella urethralis.

Studies of 31 strains of Moraxella urethralis have shown that 20 of them are competent for genetic transformation. This finding has led to the development of transformation assays for identification of newly isolated strains of this organism. Crude deoxyribonucleic acid (DNA) samples from all strains of M. urethralis readily transform auxotrophic mutants of competent strains to prototrophy, whereas DNA samples from unrelated bacteria such as Acinetobacter, Moraxella, and Neisseria species uniformly fail to elicit positive transformation of mutant tester strains. One of the competent strains of M. urethralis investigated is a naturally occurring mutant defective in its ability to utilize citrate as a carbon and energy source. DNA samples from 29 of the 30 remaining strains of utilization; the one nonreacting strain is citrate negative and probably possesses the same genetic lesion as the citrate-negative mutant. Three organisms originally identified as strains of M. urethralis, because of their phenotypic properties, are probably incorrectly designated, since DNA samples from these strains failed to transform any of the tester mutant strains used in the present study. The transformation assay for M. urethralis is very simple and can be performed readily in a clinical laboratory. The entire procedure can be carried out in less than 24 h.

Identification of Neisseria gonorrhoeae by genetic transformation: a clinical laboratory evaluation.

Transformation of a Neisseria gonorrhoeae auxotroph (uracil and arginine deficient) to prototrophy was attempted with wild-type deoxyribonucleic acid from 71 random clinical N. gonorrhoeae cultures. Of these 71 cultures, 97.1% transformed the nutritionally deficient mutant to prototrophy. The procedure was reliable and economical and offered several distinct advantages over other methods used for the confirmation of N. gonorrhoeae.

Genetic Transformation as a tool for detection of Neisseria gonorrhoeae.

A rapid method for the detection of Neisseria gonorrhoeae, making use of the ability of deoxyribonucleic acid samples from clinically isolated strains of this organism to transform nutritional mutants of a particular strain of N. gonorrhoeae, has been described. In addition to using isolated cultures, transforming deoxyribonucleic acid can be obtained directly from the material that adheres to swabs of the cervix or the urethra. The time interval for transfer of swabs to the diagnostic laboratory is not a significant factor. It is not necessary to use pure cultures on primary isolation plates to obtain definitive results. Nongonorrhoeae neisserias, as well as a large variety of commonly encountered unrelated bacteria, do not react or interfere in the transformation assay when using one of the mutant strains under a standardized set of conditions. The entire assay can be completed in less than 24 h. It has also been shown that type T4 cells of the strain of N. gonorrhoeae employed in the present study are competent for genetic transformation, although type T4 cells are transformed at a significantly lower frequency than are type T2 cells of the same strain.