Genetic Studies of Sulfadiazine-resistant and Methionine-requiring \u3cem\u3eNeisseria\u3c/em\u3e Isolated From Clinical Material

Deoxyribonucleate (DNA) preparations were extracted from Neisseria meningitidis (four isolates from spinal fluid and blood) and N. gonorrhoeae strains, all of which were resistant to sulfadiazine upon primary isolation. These DNA preparations, together with others from in vitro mutants of N. meningitidis and N. perflava, were examined in transformation tests by using as recipient a drug-susceptible strain of N. meningitidis (Ne 15 Sul-s Met+) which was able to grow in a methionine-free defined medium. The sulfadiazine resistance typical of each donor was introduced into the uniform constitution of this recipient. Production of p-aminobenzoic acid was not significantly altered thereby. Transformants elicited by DNA from the N. meningitidis clinical isolates were resistant to at least 200 μg of sulfadiazine/ml, and did not show a requirement for methionine (Sul-r Met+). DNA from six strains of N. gonorrhoeae, which were isolated during the period of therapeutic use of sulfonamides, conveyed lower degrees of resistance and, invariably, a concurrent methionine requirement (Sul-r/Met−). The requirement of these transformants, and that of in vitro mutants selected on sulfadiazine-agar, was satisfied by methionine, but not by vitamin B12, homocysteine, cystathionine, homoserine, or cysteine. Sul-r Met+ and Sul-r/Met− loci could coexist in the same genome, but were segregated during transformation. On the other hand, the dual Sul-r/Met− properties were not separated by recombination, but were eliminated together. DNA from various Sul-r/Met− clones tested against recipients having nonidentical Sul-r/Met− mutant sites yielded Sul-s Met+ transformants. The met locus involved is genetically complex, and will be a valuable tool for studies of genetic fine structure of members of Neisseria, and of genetic homology between species

Isolation of non-tuberculous mycobacteria from pastoral ecosystems of Uganda: Public Health significance

<p>Abstract</p> <p>Background</p> <p>The importance of non-tuberculous mycobacteria (NTM) infections in humans and animals in sub-Saharan Africa at the human-environment-livestock-wildlife interface has recently received increased attention. NTM are environmental opportunistic pathogens of humans and animals. Recent studies in pastoral ecosystems of Uganda detected NTM in humans with cervical lymphadenitis and cattle with lesions compatible with bovine tuberculosis. However, little is known about the source of these mycobacteria in Uganda. The aim of this study was to isolate and identify NTM in the environment of pastoral communities in Uganda, as well as assess the potential risk factors and the public health significance of NTM in these ecosystems.</p> <p>Method</p> <p>A total of 310 samples (soil, water and faecal from cattle and pigs) were examined for mycobacteria. Isolates were identified by the INNO-Lipa test and by 16S rDNA sequencing. Additionally, a questionnaire survey involving 231 pastoralists was conducted during sample collection. Data were analysed using descriptive statistics followed by a multivariable logistic regression analysis.</p> <p>Results</p> <p>Forty-eight isolates of NTM were detected; 25.3% of soil samples, 11.8% of water and 9.1% from animal faecal samples contained mycobacteria. Soils around water sources were the most contaminated with NTM (29.8%). Of these samples, <it>M. fortuitum-peregrinum </it>complex, <it>M. avium </it>complex, <it>M. gordonae</it>, and <it>M. nonchromogenicum </it>were the most frequently detected mycobacteria. Drinking untreated compared to treated water (OR = 33), use of valley dam versus stream water for drinking and other domestic use (OR = 20), sharing of water sources with wild primates compared to antelopes (OR = 4.6), sharing of water sources with domestic animals (OR = 5.3), and close contact with cattle or other domestic animals (OR = 13.8) were the most plausible risk factors for humans to come in contact with NTM in the environment.</p> <p>Conclusions</p> <p>The study detected a wide range of potentially pathogenic NTM from the environment around the pastoral communities in Uganda. Drinking untreated water and living in close contact with cattle or other domestic animals may be risk factors associated with the possibility of humans and animals acquiring NTM infections from these ecosystems.</p

Whole genome sequencing reveals mycobacterial microevolution among concurrent isolates from sputum and blood in HIV infected TB patients

YesBackground In the context of advanced immunosuppression, M. tuberculosis is known to cause detectable mycobacteremia. However, little is known about the intra-patient mycobacterial microevolution and the direction of seeding between the sputum and blood compartments. Methods From a diagnostic study of HIV-infected TB patients, 51 pairs of concurrent blood and sputum M. tuberculosis isolates from the same patient were available. In a previous analysis, we identified a subset with genotypic concordance, based on spoligotyping and 24 locus MIRU-VNTR. These paired isolates with identical genotypes were analyzed by whole genome sequencing and phylogenetic analysis. Results Of the 25 concordant pairs (49 % of the 51 paired isolates), 15 (60 %) remained viable for extraction of high quality DNA for whole genome sequencing. Two patient pairs were excluded due to poor quality sequence reads. The median CD4 cell count was 32 (IQR; 16–101)/mm3 and ten (77 %) patients were on ART. No drug resistance mutations were identified in any of the sequences analyzed. Three (23.1 %) of 13 patients had SNPs separating paired isolates from blood and sputum compartments, indicating evidence of microevolution. Using a phylogenetic approach to identify the ancestral compartment, in two (15 %) patients the blood isolate was ancestral to the sputum isolate, in one (8 %) it was the opposite, and ten (77 %) of the pairs were identical. Conclusions Among HIV-infected patients with poor cellular immunity, infection with multiple strains of M. tuberculosis was found in half of the patients. In those patients with identical strains, whole genome sequencing indicated that M. tuberculosis intra-patient microevolution does occur in a few patients, yet did not reveal a consistent direction of spread between sputum and blood. This suggests that these compartments are highly connected and potentially seed each other repeatedly