Whole genome sequencing (WGS) has enabled the development of new approaches to track Mycobacterium tuberculosis (Mtb) transmission between tuberculosis (TB) cases but its utility may be challenged by the discovery that Mtb diversifies within hosts. Nevertheless, there is limited data on the presence and degree of within-host evolution. We profiled a well-documented Mtb transmission cluster with three pulmonary TB cases to investigate within-host evolution and describe its impact on recent transmission estimates. We used deep sequencing to track minority allele frequencies (<50·0% abundance) during transmission and standard treatment. Pre-treatment (n = 3) and serial samples collected over 2 months of antibiotic treatment (n = 16) from all three cases were analysed. Consistent with the epidemiological data, zero fixed SNP separated all genomes. However, we identified six subclones between the three cases with an allele frequency ranging from 35·0% to 100·0% across sampling intervals. Five subclones were identified within the index case pre-treatment and shared with one secondary case, while only the dominant clone was observed in the other secondary case. By tracking the frequency of these heterogeneous alleles over the two-month therapy, we observed distinct signatures of drift and negative selection, but limited evidence for de novo mutations, even under drug pressure. We document within-host Mtb diversity in an index case, which led to transmission of minority alleles to a secondary case. Incorporating data on heterogeneous alleles may refine our understanding of Mtb transmission dynamics. However, more evidence is needed on the role of transmission bottleneck on observed heterogeneity between cases.

We used whole-genome sequencing to investigate a tuberculosis outbreak involving U.S.-born persons in the prison system and both U.S.- and foreign-born persons in the community in Florida over a 7-year period (2009–2015). Genotyping by spacer oligonucleotide typing and 24-locus mycobacterial interspersed repetitive unit-variable number tandem repeat suggested that the outbreak might be clonal in origin. However, contact tracing could not link the two populations. Through a multidisciplinary approach, we showed that the cluster involved distinct bacterial transmission networks segregated by country of birth. The source strain is of foreign origin and circulated in the local Florida community for more than 20 years before introduction into the prison system. We also identified novel transmission links involving foreign and U.S.-born cases not discovered during contact investigation. Our data highlight the potential for spread of strains originating from outside the United States into U.S. “high-risk” populations, such as prisoners, with subsequent movement back to the general community.

Background: Typhoid fever caused by Salmonella enterica serovar Typhi (S. typhi) continues to pose a significant risk to public health in developing countries, including Pakistan. This study investigated the epidemiological factors linked to suspected and confirmed S. typhi infections in Peshawar’s hospital population. Methodology: A total of 5735 blood samples of patients with suspected enteric fever were collected from September 2022 to November 2023. S. typhi infection was confirmed using microbiological culture of blood samples, biochemical-based tests, and DNA-sequencing methods. Drug sensitivity testing on cultures was conducted as per the CLSI guidelines. Chi-square tests were used to analyze the clinical and epidemiologic characteristics of 5735 samples stratified by S. typhi infection status, and risk factors were assessed by applying logistic regression models to estimate odds ratios (ORs). Results: The number of confirmed typhoid fever cases in this hospital-based study population was 691 (/5735, 12.0%), more prevalent in males (447/3235 13.8%) and children (0–11 years) (429/2747, 15.6%). Compared to children, the risk of S. typhi infection was lower in adolescence (adjusted OR = 0.52; 95% CI: 0.42–0.66), adulthood (19–59 years; aOR = 0.30; 95% CI: 0.25–0.38), and older adulthood (aOR = 0.08; 95% CI: 0.04–0.18) (p < 0.001). Compared to males, the risk of S. typhi infection was lower in females (aOR = 0.67; 95% CI = 0.56–0.80; p = 0.002). Living in a rural residence (compared to urban) was associated with a higher risk of infection (aOR = 1.38; 95% CI: 1.16–1.63; p = 0.001), while access to a groundwater source (compared to municipal water supply) led to a lower risk (aOR = 0.56; 95% CI: 0.43–0.73; p = 0.002). Vaccination demonstrated a robust protective effect (aOR = 0.069; 95% CI = 0.04–0.11, p = 0.002). For those with typhoid infections, clinical biomarker analysis revealed the presence of leucopenia (65/691, 9.4%), thrombocytopenia (130/691, 18.8%), and elevated alanine aminotransferase (ALT) (402/691, 58.2%) and C-reactive protein (CRP) (690/691, 99.9%) levels. Worryingly, among the positive S. typhi isolates, there was a high prevalence of drug resistance (653/691), including multidrug-resistant (MDR 82/691, 11.9%) and extensively drug-resistant types (XDR, 571/691, 82.6%). Conclusions: This study highlights the importance of age, sex, locality, water source, and vaccination status in shaping the epidemiological landscape of S. typhi in the Peshawar district. It implies that expanding vaccination coverage to the broader population of Khyber Pakhtunkhwa province, particularly in the district of Peshawar, would be beneficial.

Tuberculosis (TB) preventive therapy (TPT) is an effective strategy to eliminate TB in low-incidence settings. Shorter TPT regimens incorporating the antimicrobial class of rifamycins are designed to improve adherence and completion rates but carry the risk of modifications to the gut microbiota. We enrolled six subjects diagnosed with latent TB infection (LTBI) who accepted to initiate TPT. We also enrolled six healthy volunteers unexposed to the rifamycins. We profiled the gut microbiota using 16S rRNA amplicon sequencing (V1-V2 region) to document the immediate effect of rifamycin-based TPT on the gut microbiota composition and tracked recovery to baseline two months after TPT. Overall, TPT accounted for 17% of the variance in gut microbial community dissimilarity. This rifamycin-based TPT induced dysbiosis was characterized by a depletion of butyrate-producing taxa (Clostridium-XIVa and Roseburia) and expansion of potentially pathogenic taxa within the Firmicutes and Proteobacteria phyla. Recovery of the gut microbial composition was incomplete two months after TPT. Robust clinical studies are necessary to comprehensively catalogue TPT-induced gut microbiota dysbiosis to inform strategies to mitigate potential long-term sequelae of this important TB control intervention.