Incidence and distribution of human leptospirosis in the Western Cape Province, South Africa (2010-2019): a retrospective study.

Introduction: leptospirosis is an emerging zoonosis of global importance. In South Africa, the infection is an underreported public health concern, with limited information on its incidence and distribution. This study investigated the incidence of human leptospirosis in Western Cape Province (WCP) between 2010 and 2019, and compared the incidence based on seasonal and demographic factors. Methods: a retrospective study was conducted with data on leptospirosis diagnoses in WCP obtained from the National Health Laboratory Services. With the provincial population sizes as the denominator, incidence of leptospirosis was estimated and expressed as cases per 100,000 population. Negative binomial regression was used to estimate the effect of sex, season, and year on the incidence of leptospirosis. Results: two hundred and fifty-four (254) cases of leptospirosis were reported between 2010 and 2019, with the highest number of cases being in 2015 and the annual incidence ranged between 0.15 and 0.66/100,000 population. Males had a higher incidence compared to females (0.55 vs. 0.25/ 100,000 population; incidence rate ratio (IRR) 2.2, 95% CI: 1.66,3.03). The 18-44 age cohort had the highest average incidence (0.56/100,000 population), while the ≤17 age cohort had the lowest incidence (0.07/100,000 population). The 18-44 (IRR 8.0, 95% CI: 4.65,15.15) and ≥45 (IRR 7.4, 95% CI: 4.17,14.17) age cohorts were more at risk of infection compared to ≤17 age cohort. Conclusion: leptospirosis is an important zoonosis in WCP disproportionately affecting males and the productive age demographic groups. These findings should enhance targeted prevention and provoke further investigation on the importance of environmental and socioeconomic factors on leptospirosis burden.

Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates.

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1 (g609a), and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.

Prognostic significance of novel katG mutations in Mycobacterium tuberculosis.

BACKGROUND: By using whole genome sequencing (WGS), researchers are beginning to understand the genetic diversity of Mycobacterium tuberculosis (MTB) and its consequences for the diagnosis of multidrug-resistant tuberculosis (MDR-TB) on a genomic scale. The Global Consortium for Drug-resistant TB Diagnostics (GCDD) conducted a genome scale variant analyses of 366 clinical MTB genomes (mostly MDR/XDR [extensively drug resistant]) from four countries in order to inform the development of rapid molecular diagnostics. This project has been extended by performing an evolutionary analysis of isoniazid (INH)-resistant isolates for prognostic purposes. METHODS: 151 (130 INHR, 21 INHS) clinical MTB isolates from India (19: 17 INHR, 2 INHS), Moldova (48: 42 INHR, 6 INHS), the Philippines (26: 20 INHR, 6 INHS), and South Africa (58: 51 INHR, 7 INHS) were included in this study. INH drug susceptibility was determined by using MGIT 960 and WHO (World Health Organization)-recommended critical concentration of 0.1 mg/L. Isolates were sequenced using PacBio RS WGS platform. A genome-wide variant analysis was conducted using a proprietary pipeline (PacDAP) developed at San Diego State University. To infer the amino acid changes in katG that confer resistance, PAML was utilized to detect sites in silico that are under positive selection. The dN/dS method was used in combination with Bayes empirical Bayes to determine sites under positive selection and Chi-Squared analysis to determine the significance of the selected sites. RESULTS: PacDAP variant analysis revealed 22 novel catalase-peroxidase (katG product) mutations. Of these, 14 were single nucleotide polymorphisms, while 8 novel mutations appeared in combination with katG S315T and/or with inhA promoter C-15T. These SNPs have not been previously reported. Additionally, 11 previously observed, but uncommon, katG mutations were also observed in these clinical isolates. These results suggest that 17 amino acids in the enzyme are under positive selective pressure; most significantly in South Africa and the Philippines. No selective pressure on codons other than 315 was observed in isolates from Moldova. Due to the low number of isolates from India, the significance of the sites under positive selection was low and no prediction for India could be made based on this study. CONCLUSIONS: Eleven of the 14 SNPs are resistance conferring, and it is believed that the remaining 8 combinatorial mutations are either compensatory in nature or, in combination with known SNPs, could increase resistance levels. Positive selection results indicate a diversifying evolutionary path to resistance more in line with long tail statistics and therefore indicate a departure from the traditional point mutation (or "hotspot") model that current molecular diagnostics are based on. Positive selection pressures indicate a future with elevated diagnostic and prognostic significance of the "long tail" (i.e., alternative mechanisms of resistance) and potentially diminishing significance of the canonical mutations (especially in South Africa and the Philippines), which could have significant future implications on narrowly targeting molecular diagnostics.

The role of Bacteroides fragilis RecQ DNA helicases in cell survival after metronidazole exposure.

The inactivation of Bacteroides fragilis genes encoding putative RecQ helicases recQ1, recQ2 and recQ3 (ORFs BF638R_3282, BF638R_3781, BF638R_3932) was used to determine whether these proteins are involved in cell survival following metronidazole exposure. The effects of the mutations on growth, cellular morphology and DNA integrity were also evaluated. Mutations in the RecQ DNA helicases caused increased sensitivity to metronidazole, with recQ1, recQ2 and recQ3 mutants being 1.32-fold, 41.88-fold and 23.18-fold more sensitive than the wild type, respectively. There was no difference in cell growth between the recQ1 and recQ3 mutants and the wild type. However, the recQ2 mutant exhibited reduced cell growth, aberrant cell division and increased pleiomorphism, with an increase in filamentous forms and chains of cells being observed using light, fluorescence and electron microscopy. There was no spontaneous accumulation of DNA single- or double-strand breaks in the recQ mutants, as compared with the wild type, during normal cell growth in the absence of metronidazole. Bacteroides fragilis RecQ DNA helicases, therefore, enhance cell survival following metronidazole damage. The abnormal cellular phenotype and growth characteristics of recQ2 mutant cells suggest that this gene, or the downstream gene of the operon in which it occurs, may be involved in cell division.

Bacteroides fragilis RecA protein overexpression causes resistance to metronidazole.

Bacteroides fragilis is a human gut commensal and an opportunistic pathogen causing anaerobic abscesses and bacteraemias which are treated with metronidazole (Mtz), a DNA damaging agent. This study examined the role of the DNA repair protein, RecA, in maintaining endogenous DNA stability and its contribution to resistance to Mtz and other DNA damaging agents. RT-PCR of B. fragilis genomic DNA showed that the recA gene was co-transcribed as an operon together with two upstream genes, putatively involved in repairing oxygen damage. A B. fragilis recA mutant was generated using targeted gene inactivation. Fluorescence microscopy using DAPI staining revealed increased numbers of mutant cells with reduced intact double-stranded DNA. Alkaline gel electrophoresis of the recA mutant DNA showed increased amounts of strand breaks under normal growth conditions, and the recA mutant also showed less spontaneous mutagenesis relative to the wild type strain. The recA mutant was sensitive to Mtz, ultraviolet light and hydrogen peroxide. A B. fragilis strain overexpressing the RecA protein exhibited increased resistance to Mtz compared to the wild type. This is the first study to show that overexpression of a DNA repair protein in B. fragilis increases Mtz resistance. This represents a novel drug resistance mechanism in this bacterium.

Overexpression of the rhamnose catabolism regulatory protein, RhaR: a novel mechanism for metronidazole resistance in Bacteroides thetaiotaomicron.

OBJECTIVES: The aim of the investigation was to use in vitro transposon mutagenesis to generate metronidazole resistance in the obligately anaerobic pathogenic bacterium Bacteroides thetaiotaomicron, and to identify the genes involved to enable investigation of potential mechanisms for the generation of metronidazole resistance. METHODS: The genes affected by the transposon insertion were identified by plasmid rescue and sequencing. Expression levels of the relevant genes were determined by semi-quantitative RNA hybridization and catabolic activity by lactate dehydrogenase/pyruvate oxidoreductase assays. RESULTS: A metronidazole-resistant mutant was isolated and the transposon insertion site was identified in an intergenic region between the rhaO and rhaR genes of the gene cluster involved in the uptake and catabolism of rhamnose. Metronidazole resistance was observed during growth in defined medium containing either rhamnose or glucose. The metronidazole-resistant mutant showed improved growth in the presence of rhamnose as compared with the wild-type parent. There was increased transcription of all genes of the rhamnose gene cluster in the presence of rhamnose and glucose, likely due to the transposon providing an additional promoter for the rhaR gene, encoding the positive transcriptional regulator of the rhamnose operon. The B. thetaiotaomicron metronidazole resistance phenotype was recreated by overexpressing the rhaR gene in the B. thetaiotaomicron wild-type parent. Both the metronidazole-resistant transposon mutant and RhaR overexpression strains displayed a phenotype of higher lactate dehydrogenase and lower pyruvate oxidoreductase activity in comparison with the parent strain during growth in rhamnose. CONCLUSIONS: These data indicate that overexpression of the rhaR gene generates metronidazole resistance in B. thetaiotaomicron.

Rhamnose catabolism in Bacteroides thetaiotaomicron is controlled by the positive transcriptional regulator RhaR.

Bacteroides thetaiotaomicron is an important human gut commensal, which also causes opportunistic infections outside this environment. It utilises a range of host and diet-related carbohydrates, including rhamnose. In this study, the rha gene cluster, required for rhamnose utilisation, was characterised by transcription analysis, gene targeted mutagenesis and enzyme assays. Growth in the presence of L-rhamnose induced transcription of all the genes of this cluster. The first five genes of the cluster, rhaKIPAO, were transcribed as an operon from a transcriptional start site upstream of rhaK, whereas the sixth gene, rhaR, was transcribed independently. Bioinformatic analysis and mutation of the rhaR gene identified it as encoding the positive transcriptional activator of rhaKIPAO. A rhaR mutant could not utilise rhamnose as the sole carbon source but grew normally on glucose. The rhaO gene encoded a lactaldehyde reductase, and a rhaO mutant produced reduced levels of L-1,2-propanediol during growth in rhamnose, indicating its contribution to rhamnose catabolism in Bacteroides thetaiotaomicron.

An AraC/XylS family transcriptional regulator homologue from Bacteroides fragilis is associated with cell survival following DNA damage.

A putative transcriptional regulator of the AraC/XylS family was identified in a genomic genebank of Bacteroides fragilis Bf-1, which partially relieved the sensitivity of Escherichia coli DNA repair mutants to the DNA-damaging agents, metronidazole and mitomycin C. A homologue of this gene with the same phenotype was identified as BF638R3281 in B. fragilis 638R. Transcription of BF638R3281 was constitutive with respect to exposure to sublethal doses of metronidazole. BF638R3281 was interrupted by single cross-over gene-specific insertion mutation, and the gene disruption was confirmed by PCR and DNA-sequencing analysis. The mutant grew more slowly than the wild type, and the mutation rendered B. fragilis more sensitive to metronidazole and mitomycin C. This indicates that the BF638R3281 gene product plays a role in the survival of B. fragilis following DNA damage by these agents.