Exploitation of the broad specificity of the membrane-bound isoenzyme of lactate dehydrogenase for direct selection of null mutants in Neisseria gonorrhoeae.

Lactic acid is readily utilized as a carbon and energy source by Neisseria gonorrhoeae. The oxidation of lactate is coupled to electron transport via a membrane-bound lactate dehydrogenase (iLDH) which is independent of pyridine nucleotide. The broad substrate specificity of iLDH endows N. gonorrhoeae with the novel ability to convert phenyllactate to L-phenylalanine via phenylpyruvate. N. gonorrhoeae ATCC 27628 typifies a class of clinical isolate whose growth is inhibited by phenylpyruvate (or L-phenylalanine). Exploiting resistance to growth inhibition by phenyllactate as a strategy of positive selection, mutant derivatives of strain ATCC 27628 lacking iLDH activity were readily obtained. These mutants are incapable of oxidizing phenyllactate, and lack the parent-strain ability to reduce c-type cytochromes in the presence of lactate, phenyllactate or 4-hydroxyphenyllactate. They retain, however, a cytoplasmic NAD(+)-linked lactate dehydrogenase (nLDH). Since the mutants retained the ability to grow on lactate as a sole source of carbon, nLDH presumably can function in an opposite-to-normal physiological direction in the absence of iLDH. This would explain the failure to isolate iLDH-deficient mutants by selection for inability to grow on lactate.

The biochemical basis for growth inhibition by L-phenylalanine in Neisseria gonorrhoeae.

Clinical isolates of Neisseria gonorrhoeae are commonly subject to growth inhibition by phenylpyruvate or by L-phenylalanine. A blockade of tyrosine biosynthesis is indicated since inhibition is reversed by either L-tyrosine or 4-hydroxyphenylpyruvate. Phenylalanine-resistant (PheR) and phenylalanine-sensitive (PheS) isolates both have a single 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase that is partially inhibited by L-phenylalanine (80%). However, PheS and PheR isolates differ in that the ratio of phenylpyruvate aminotransferase to 4-hydroxyphenylpyruvate aminotransferase is distinctly greater in PheS isolates than in PheR isolates. A mechanism for growth inhibition is proposed in which phenylalanine exerts two interactive effects. (i) Phenylalanine decreases precursor flow to 4-hydroxyphenylpyruvate through its controlling effect upon DAHP synthase; and (ii) phenylalanine is largely transaminated to phenylpyruvate, which saturates both aminotransferases, preventing transamination of an already limited supply of 4-hydroxyphenylpyruvate to L-tyrosine.

The broad-specificity, membrane-bound lactate dehydrogenase of Neisseria gonorrhoeae: ties to aromatic metabolism.

Neisseria gonorrhoeae can convert phenyllactate (PL) to phenylalanine and 4-hydroxyphenyllactate (HPL) to tyrosine. This was demonstrated by nutritional and physiological approaches. The enzymic basis for this unusual ability was shown to be the broad specificity of a particulate, unidirectional, pyridine-nucleotide-independent lactate dehydrogenase. This enzyme, denoted [iLDH], has been implicated in a pathogenic mechanism whereby host-derived lactate is linked to increased gonococcal oxygen consumption and electron transport. A similar role for HPL, a metabolite available in human host tissues, may provide a selective basis to explain evolution of broadened [iLDH] specificity in Neisseria. The interplay between aromatic metabolism and [iLDH] suggests new approaches for manipulating the host-pathogen relationship.

Enzymic arrangement and allosteric regulation of the aromatic amino acid pathway in Neisseria gonorrhoeae.

The pathway construction and allosteric regulation of phenylalanine and tyrosine biosynthesis was examined in Neisseria gonorrhoeae. A single 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase enzyme sensitive to feedback inhibition by L-phenylalanine was found. Chorismate mutase and prephenate dehydratase appear to co-exist as catalytic components of a bifunctional enzyme, known to be present in related genera. The latter enzyme activities were both feedback inhibited by L-phenylalanine. Prephenate dehydratase was strongly activated by L-tyrosine. NAD+-linked prephenate dehydrogenase and arogenate dehydrogenase activities coeluted following ion-exchange chromatography, suggesting their identity as catalytic properties of a single broad-specificity cyclohexadienyl dehydrogenase. Each dehydrogenase activity was inhibited by 4-hydroxyphenylpyruvate, but not by L-tyrosine. Two aromatic aminotransferases were resolved, one preferring the L-phenylalanine:2-ketoglutarate substrate combination and the other preferring the L-tyrosine: 2-ketoglutarate substrate combination. Each aminotransferase was also able to transaminate prephenate. The overall picture of regulation is one in which L-tyrosine modulates L-phenylalanine synthesis via activation of prephenate dehydratase. L-Phenylalanine in turn regulates early-pathway flow through inhibition of DAHP synthase. The recent phylogenetic positioning of N. gonorrhoeae makes it a key reference organism for emerging interpretations about aromatic-pathway evolution.

Zygomycosis. Report of four cases with formation of chlamydoconidia in tissue.

The authors describe spheric to ovoid chlamydoconidia and mucoraceous hyphae in tissues from four patients, two with cutaneous and two with pulmonary zygomycosis. The diagnosis in each case was confirmed by immunofluorescence staining and the presence of characteristic hyphae in tissue. It is important that these conidia be recognized, because they can easily be mistaken for other fungi, nematode ova, or other microorganisms in tissue sections, thereby resulting in the potential for misdiagnosis.

Growth inhibition of Neisseria gonorrhoeae isolates by L-phenylalanine and its analogues in defined media.

Growth inhibition by phenylalanine (0.25 mmol/L in defined agar media) was present in about 1% of over 1000 clinical isolates of Neisseria gonorrhoeae isolates tested. Turbidometry of several phenylalanine-sensitive isolates showed that their growth rates decreased in proportion to phenylalanine concentrations up to about 1 mmol/L. The growth rate was unaffected if 0.04 mmol/L tyrosine was also present. The phenylalanine analogue DL-3-fluorophenylalanine inhibited the growth of all 23 isolates further tested on agar. This inhibition was depressed by phenylalanine in all 17 phenylalanine-resistant isolates. Phenylalanine plus tyrosine were required to derepress the analogue inhibition in the other six phenylalanine-sensitive isolates. Phenylalanine-sensitive isolates may have a defect in aromatic amino acid synthesis, not involving auxotrophy, but manifested through regulation of the pathways. Phenylalanine effectively repressed tyrosine and phenylalanine synthesis. In 125 isolates including 85 beta-lactamase producers (PPNG) and 32 phenylalanine-sensitive isolates, phenylalanine inhibited 63.2% of 38 PPNG isolates carrying the 3.2 megadalton (Md) plasmid, but only one of 47 PPNG isolates carrying the 4.5 Md plasmid. PPNG isolates are most often of the proline, ornithine, or nonrequiring auxotypes. Phenylalanine sensitivity did not appear to be auxotype dependent.

Growth responses of Neisseria gonorrhoeae auxotypes to required amino acids and bases in liquid medium.

Neisseria gonorrhoeae strains can be grouped or differentiated (auxotyped) by their requirements for none, or any one or more of proline, uracil, hypoxanthine, and citrulline or ornithine. Most strains were readily assessed because they responded with growth or no growth on each defined auxanographic medium. Other strains gave indeterminate responses on agar and the reasons were not obvious. Liquid growth studies for quantifying the usual responses showed that yields of appropriate N. gonorrhoeae auxotrophs were proportional to replacement concentrations of any one of these amino acids or bases, of methionine, or of cysteine plus cystine. This type of response, where log growth rates and lag times were unaffected, is proposed as the basis for defining (simple) auxotrophy in gonococci. The formula of the defined medium was improved by increasing proline, uracil, and hypoxanthine beyond limiting concentrations, and decreasing citrulline or ornithine, and cysteine plus cystine. Fatty acid--free bovine albumin was used to ensure homogeneous growth in liquid media. In agar, it was superior to starch for the nonnutritive protective effect required by many strains.

Association between the auxogroup of Neisseria gonorrhoeae and the minimal inhibitory concentration of penicillin.

The auxogroups and the minimal inhibitory concentrations (MICs) of penicillin for 885 recently isolated strains of Neisseria gonorrhoeae were determined. Auxogroups were established according to the growth requirements of the strains for proline (Pro-), uracil (Ura-), hypoxanthine (Hyx-), citrulline (Cit-), or citrulline replaceable by ornithine (Orn-). The seven most common auxogroups studied were: Pro-Cit-Ura- (38.8%), Orn-Hyx-Ura- (24.1%), nonrequiring (NR) strains (13.4%), Pro- (9.7%), Orn- (5.2%), Cit-Hyx-Ura- (4.2%), and Pro-Onr-Hyx-Ura- (2.0%). The distribution of MICs of penicillin for the complete set of 885 strains had a bimodal distribution, with a minor peak at 0.01 microgram of penicillin/ml and a major one at 0.16 microgram/ml. The auxogroups, however, had different patterns of distribution of MICs of penicillin. Five patterns of distribution were found among eight auxogroups identified. (1) Orn- and NR auxogroups showed a broad distribution of MICs, with peak at 0.08 microgram/ml. (2) Of the Cit-Hyx-Ura- and Orn-Hyx-Ura- auxogroups, greater than 98% of strains were susceptible to less than or equal to 0.04 microgram of penicillin/ml. (3) All strains of the Pro-Orn-Hyx-Ura- and Pro-Cit-Hyx-Ura- auxogroups were susceptible to less than or equal to 0.08 microgram of penicillin/ml. (4) More than 95% of strains of the Pro-Cit-Ura- auxogroup required greater than of equal to 0.08 microgram of penicillin/ml for inhibition of growth. (5) The Pro- auxogroup showed a bimodal distribution, with a major peak at 0.01 microgram of penicillin/ml and a minor one at 0.32 microgram/ml.

Silver-resistant Enterobacteriaceae from hospital patients.

The inclusion of agar medium containing 0.5 mM AgNO3 in the hospital laboratory replicating system for routine antibiotic-susceptibility determinations resulted in identification of species of Enterobacteriaceae (Escherichia coli, Enterobacter cloacae, Klebsiella pneumoniae, Proteus mirabilis, and Citrobacter freundii) with silver resistance. Since the study began in October, 1975, 11 in-hospital patients receiving silver sulfadiazine for burn wound prophylaxis have yielded silver-resistant bacteria from their infected burns. During this treatment routine burn-site cultures from these patients yielded 230 isolates of Enterobacteriaceae, including 211 which were sulfonamide-resistant, 97 of which were also silver-resistant, and 38 of which were untested for silver resistance. Seven silver-resistant but sulfonamide-sensitive isolates were incidentally recovered from respiratory specimens from four nonburn patients with silver tracheostomy tubes, one silver-resistant sulfonamide-sensitive isolate was recovered from a small infected burn on the foot of an Emergency Room patient. Previous treatment of this burn was unknown. Representative AgNO3-resistant E. coli isolates from four patients were serologically untypable. Serotyping of representative isolates of K. pneumoniae showed a diversity of types except from two patients who had been in the same ward at the same time.

Auxanographic grouping and typing of Neisseria gonorrhoeae.

Nearly 96% of 1297 Neisseria gonorrhoeae isolates in Hamilton were assigned to six major auxanographic groups (non-requiring or NR, Pro-, Orn-, Pro-Cit-Ura-, Orn-Ura-Hyx-, Cit-Ura-Hyx-) as established by requirements for none, or any one or more of proline, uracil, hypoxanthine, citrulline (Cit-), or citrulline replaceable by ornithine (Orn-) on chemically defined medium modified from Catlin (1973). Seven other groups, and strains not growing, accounted for 4.2%. The most common groups were Orn-Ura-Hyx- (25.6%) and Pro-Cit-Ura (31.8%). This latter group has not been previously described. These "Pro Cit/Orn Ura Hyx" criteria were among the most unequivocal to interpret; with rare exceptions for proline, a requirement was shown by absence of growth at any time in the zones of inoculation from a replicator. For some strains, some of the possible additional requirements (leucine, valine, isoleucine, glutamine, threonine, serine, histidine, etc.) could be less readily ascertained, because an occasional manifestation was to reduce the amount of growth, or to slow down the rate of growth, compared to complete medium. The first four of the six groups were the least fastidious, in that few strains had any additional requirements. About 2% of strains were inhibited by 0.25 mM phenylalanine.

A case of pulmonary veno-occlusive disease respondong to treatment with azathioprine.

Histological features of a lung biopsy specimen from a 46-year-old woman showed all the characteristics described in veno-occlusive disease. The clinical features, however, were distinctive in that in addition to the lung involvement there was alopecia, digital vasculitic ulcers, Raynaud's phenomenon, polyarthritis, and muscle weakness. Treatment with azathioprine resulted in a progressive improvement in her condition. It is suggested that pulmonary small vein occlusion may occur as a pattern of tissue response in more than one situation and that is sometimes more amenable to therapy than has been previously reported.

The combined use of betamethasone valerate and sodium cromoglycate in the treatment of asthma.

A double-blind comparison of betamethasone valerate, sodium cromoglycate and the combination of these two treatments was carried out in twenty-two adult patients with asthma. Regular fortnightly assessments were made in the clinic throughout the study and adrenal function was monitored and found to be normal. All patients measured their peak expiratory flow rates in the morning and evening and monitored their symptoms daily on a record card as well as recording bronchodilator usage. Assessment using these parameters indicated that treatment with betamethasone valerate compared with sodium cromoglycate resulted in an improvement in the patients' asthma which was stitistically significant (P less than 0-001). Overall the combined treatment produced a better response than sodium cromoglycate (P less than 0-02) but a poorer response compared with the steroid aerosol given alone (P greater than 0-05). In only two patients was the response to the combined therapy significantly greater than to either drug given alone.