Isolation and identification of nontuberculous mycobacteria from foods as possible exposure sources.

A variety of foods collected from local supermarkets and produce stands were examined as possible sources of nontuberculous mycobacterial exposure. Food samples were combined with sterile ultrapure water and manually shaken. To remove large particles, the suspensions were filtered through a sterile strainer, centrifuged, and the supernatants were discarded. The food pellets were stored at -75 degrees C. The pellets were treated with either oxalic acid or sodium hydroxide-sodium citrate solutions to reduce contamination by nonmycobacterial organisms. Decontaminated pellets were cultured on both Middlebrook 7H10C agar and Middlebrook 7H10C agar with supplemental malachite green. Plates were observed for growth at 2 and 8 weeks. Isolates demonstrating acid-fastness were identified to species using polymerase chain reaction and restriction enzyme analysis. Nontuberculous mycobacteria (NTM) were recovered from 25 of 121 foods. Six different species of NTM were isolated, the most predominant being Mycobacterium avium.

Efficient assessment of neuropathic pain drugs in patients with small fiber sensory neuropathies.

We sought to develop an enrichment crossover study design that would allow us to efficiently evaluate and compare promising candidate neuropathic pain drugs. We evaluated the efficacy of gabapentin or tramadol vs. active placebo (diphenhydramine) in subjects with biopsy-proven painful idiopathic small fiber neuropathy (SFN) who were self-reported gabapentin responders. Eligible subjects entered two single blind run-in phases. In the first phase (Period A), subjects were treated with single blinded gabapentin at their prestudy dose followed by a second run-in phase (Period B) in which they were treated with diphenhydramine active placebo. Subjects with >or=3 pain and a >or=30% increase in pain intensity in Period B compared to Period A were then randomized to a double-blind three period cross over trial of gabapentin at pre study dosage, tramadol 50mg QID and diphenhydramine 50mgqhs. Of the 59 subjects enrolled, 41 subjects were excluded: Twenty-three had an insufficient rise in pain intensity in Period B; eight had skin biopsies that did not confirm SFN. Eighteen subjects were randomized into the double-blind, crossover phase. There was a significant treatment effect of gabapentin vs. diphenhydramine (p=0.001) and tramadol vs. diphenhydramine (p=0.018) by the before-bed daily pain score averaged over the final 7 days of each treatment period. We conclude that gabapentin and tramadol were effective in the treatment of painful SFN and that this experimental enrichment paradigm is attractive to screen potential neuropathic pain compounds for efficacy in proof-of-concept studies.

Tuberculin activity of mycobacterial fractions obtained by chromatography.

Commercial purified protein derivatives (PPD), old tuberculin (OT), the bacillary extract, and the culture filtrate of Mycobacterium tuberculosis H37Ra were submitted to Sephadex G-25 and diethylaminoethyl (DEAE)-cellulose chromatography. The ability of the fractions obtained to elicit delayed dermal hypersensitivity in M. tuberculosis H37Ra-infected guinea pigs was studied. Skin tests with Sephadex fractions in M. tuberculosis H37Ra-infected guinea pigs showed that the tuberculin activity was localized in the first fraction. All other Sephadex fractions were nonessential and nonspecifically irritating. Fractions from chromatography of Sephadex G-25 fraction 1 on DEAE-cellulose columns showed that all but the first were able to elicit delayed hypersensitivity reactions. There was a variability in the capacity to elicit the tuberculin reaction according to the fraction injected and the stage of tuberculous infection in guinea pigs. Compared to the others, the seven lots of commercial PPD were variable in composition and content. They contained both essential and nonessential materials for the tuberculin reaction. Sephadex fraction 1 would appear to be a better tuberculin as it excludes nonessential nonspecifically irritating elements and contains the complement able to elicit the tuberculin reaction. Its methodological simplicity would be economically advantageous.

Antigens of mycobacterial cell walls.

Three antigenic fractions from the cell walls of eight strains of mycobacteria were studied. Isolation and purification of these antigens were effected by enzymatic digestions, differential and sucrose gradient centrifugations, dialyses, and column chromatography. Two of the fractions were termed cell wall tuberculins (CWT-1, solubilized with lipase; CWT-2, solubilized with lysozyme); the third was termed "C" (cross-reacting) antigen. All appeared to be lipopolysaccharides. The CWT antigens, as compared with purified protein derivatives (human), were relatively species (group)-specific in both double immunodiffusion and guinea pig skin tests; in the latter, the reactions resembled those of delayed hypersensitivity. The C antigens reacted heterologously in double immunodiffusion and skin tests; the latter were the "immediate" type of reaction.

A co-operative numerical analysis of Mycobacterium gastri, Mycobacterium kansasii and Mycobacterium marinum.

A co-operative taxonomic study has been performed on slowly growing photochromogenic mycobacteria (Runyon Group I) and closely related organisms. Phenetic data on 54 strains, studied in seven laboratories, were collected and analysed by numerical taxonomic methods. Immunological properties and phage susceptibility patterns were analysed independently to establish correlation with numerical classification. Mycobacterium gastri, M. kansasii and M. marinum appeared as distinct well-defined clusters and the serological and phage data supported the resolution of these three species. A table of definitive properties is presented. Two strains each of M. simiae and M. asiaticum formed a loose cluster which was clearly separated from the previously mentioned three species; the small number of strains examined precluded the establishment of a list of definitive properties of these two species. It is concluded that the Runyon Groups, which provided a practical though arbitrary basis for establishment of a series of co-operative studies, have served their purpose and should now be supplanted by classification and nomenclature based on species.

Comparison of large restriction fragments of Mycobacterium avium isolates recovered from AIDS and non-AIDS patients with those of isolates from potable water.

We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of M. avium isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of M. avium infections.

Phage typing of Staphylococcus intermedius.

Staphylococcus intermedius, a coagulase-positive staphylococcal species, is a common canine pathogen and a rare human wound pathogen. A total of 145 strains of S. intermedius (ATCC 29663, 4 reference strains, 4 human isolates, 44 canine infection isolates, and 92 isolates from canine gingiva) were screened for lysogenic phage by a modified Fisk method. Nineteen phage preparations were prepared for preliminary typing experiments. Lytic activity was observed on 93 of 145 (64.1%) isolates, yielding 44 lytic patterns with individual strains susceptible to one or more phages. Five phages lysed only a single strain, but lytic patterns varied from 1 to 11 lytic phages per isolate. A distinct lytic pattern did not separate canine or human wound isolates from canine gingival isolates. All human wound isolates fell into the two most common canine gingival or wound patterns; the single human nasopharyngeal isolate was not lysed by any phage. Twenty-two of 44 (55%) canine wound isolates and 65 of 92 (71%) gingival isolates yielded lytic patterns. Lysogenic phages are common in S. intermedius. This preliminary study suggests that phage typing may be a useful tool in distinguishing epidemiologically related strains.

Molecular technique for rapid identification of mycobacteria.

Identification of mycobacteria through conventional microbiological methods is cumbersome and time-consuming. Recently we have developed a novel bacterial identification method to accurately and rapidly identify different mycobacteria directly from water and clinical isolates. The method utilizes the PCR to amplify a portion of the small subunit rRNA from mycobacteria. The 5' PCR primer has a fluorescent label to allow detection of the amplified product. The PCR product is digested with restriction endonucleases, and an automated DNA sequencer is employed to determine the size of the labeled restriction fragments. Since the PCR product is labeled only at the 5' end, the analysis identifies only the restriction fragment proximal to the 5' end. Each mycobacterial species has a unique 5' restriction fragment length for each specific endonuclease. However, frequently the 5' restriction fragments from different species have similar or identical lengths for a given endonuclease. A set of judiciously chosen restriction enzymes produces a unique set of fragments for each species, providing us with an identification signature. Using this method, we produced a library of 5' restriction fragment sizes corresponding to different clinically important mycobacteria. We have characterized mycobacterial isolates which had been previously identified by biochemical test and/or nucleic acid probes. An analysis of these data demonstrates that this protocol is effective in identifying 13 different mycobacterial species accurately. This protocol has the potential of rapidly (less than 36 h) identifying mycobacterial species directly from clinical specimens. In addition, this protocol is accurate, sensitive, and capable of identifying multiple organisms in a single sample.

PCR comparison of Mycobacterium avium isolates obtained from patients and foods.

Mycobacterium avium is a cause of disseminated disease in AIDS patients. A need for a better understanding of possible sources and routes of transmission of this organism has arisen. This study utilized a PCR typing method designed to amplify DNA segments located between the insertion sequences IS1245 and IS1311 to compare levels of relatedness of M. avium isolates found in patients and foods. Twenty-five of 121 food samples yielded 29 mycobacterial isolates, of which 12 were M. avium. Twelve food and 103 clinical M. avium isolates were tested. A clinical isolate was found to be identical to a food isolate, and close relationships were found between two patient isolates and two food isolates. Relatedness between food isolates and patient isolates suggests the possibility that food is a potential source of M. avium infection. This study demonstrates a rapid, inexpensive method for typing M. avium, possibly replacing pulsed-field gel electrophoresis.